Over the years, thousands of scientists have published their scientific articles or posters where they’ve used an Ambion® product to get to the data presented. Below you’ll find some of these publications, conveniently sorted by the specific Ambion® product used. Learn more about the Ambion® Publication Rewards Program > |
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Publications Submitted By Your Colleagues
Ambion® RNAlater® Tissue Collection: RNA Stabilization Solution is an aqueous, nontoxic tissue storage reagent that rapidly permeates tissues to stabilize and protect cellular RNA
2012
RNA-Sequencing as Useful Screening Tool in the Combat against the Misuse of Anabolic Agents
Authors: I Riedmaier, V Benes, J Blake, N Bretschneider, C Zinser, C Becker, HHD Meyer, MW Pfaffl
The abuse of anabolic substances in animal husbandry is forbidden within the EU and well controlled by detecting substance residues in different matrices. The application of newly designed drugs or substance cocktails represents big problems. Therefore developing sensitive test methods is important. The analysis of physiological changes caused by the use of anabolic agents on the molecular level, for example, by quantifying gene expression response, is a new approach to develop such screening methods. A novel technology for holistic gene expression analysis is RNA sequencing. In this study, the potential of this high-throughput method for the identification of biomarkers was evaluated. The effect of trenbolone acetate plus estradiol on gene expression in liver from Nguni heifers was analyzed with RNA sequencing.
2012
Efficient and robust RNA-seq process for cultured bacteria and complex community transcriptomes
Authors: G Giannoukos, DM Ciulla, K Huang, BJ Haas, J Izard, JZ Levin, J Livny, AM Earl, D Gevers, DV Ward, C Nusbaum, BW Birren, A Gnirke
We have developed a process for transcriptome analysis of bacterial communities that accommodates both intact and fragmented starting RNA and combines efficient rRNA removal with strand-specific RNA-seq. We applied this approach to an RNA mixture derived from three diverse cultured bacterial species and to RNA isolated from clinical stool samples. The resulting expression profiles were highly reproducible, enriched up to 40-fold for non-rRNA transcripts, and correlated well with profiles representing undepleted total RNA.
2012
Transcriptomes of Mouse Olfactory Epithelium Reveal Sexual Differences in Odorant Detection
Authors: M-S Shiao, A Y-F Chang, B-Y Liao, Y-H Ching, M-Y J Lu, SM Chen, W-H Li
To sense numerous odorants and chemicals, animals have evolved a large number of olfactory receptor genes (Olfrs) in their genome. In particular, the house mouse has ∼1,100 genes in the Olfr gene family. This makes the mouse a good model organism to study Olfr genes and olfaction-related genes.
2012
Authors: Y Li, T Taketo, YF Lau
Gonadal sex differentiation is an important developmental process, in which a bipotential primordial gonad undergoes two distinct pathways, i.e., testicular and ovarian differentiation, dependent on its genetic sex. Techniques of isolating fetal gonads at various developmental stages are valuable for studies on the molecular events involved in cell-fate determination, sex-specific somatic and germ-cell differentiation and structural organization. Here we describe various procedures for isolation of embryonic gonads at different developmental stages from embryos of timed-pregnant mice. The isolated fetal gonads can be used for a variety of studies, such as organ culture, gene and protein expression.
2012
A Study in Vivo of the Effects of a Static Compressive Load on the Proximal Tibial Physis in Rabbits
Authors: AD Bries, DS Weiner, R Jacquet, MJ Adamczyk, MA Morscher, E Lowder, MJ Askew, RP Steiner, WI Horne, WJ Landis
The effect of compression on the physis is generally defined by the Hueter-Volkmann principle, in which decreased linear growth of the physis results from increased compression. This investigation examined whether mechanically induced compression of rabbit physes causes changes in gene expression, cells, and extracellular components that promote physeal resilience and strength (type-II collagen and aggrecan) and cartilage hypertrophy (type-X collagen and matrix metalloprotease-13).
2011
Authors: B Kasztelewicz, I Jankowska, J Pawłowska, J Teisseyre, K Dzierżanowska-Fangrat
Immunosuppressed pediatric transplant recipients are at risk of developing Epstein–Barr virus (EBV)-associated complications (such as post-transplant lymphoproliferative disorders). Monitoring of the EBV DNA level in blood alone has a low predictive value for the post-transplant course of EBV infection and its complications. Therefore, additional prognostic markers are widely sought. The study aim was to analyze EBV gene expression patterns and LMP1 polymorphism in relation to EBV DNA levels in pediatric liver transplant recipients. EBV load measurement, LMP1 variant, and gene expression analysis were performed in collected prospectively multiple blood samples from 30 patients.
2008
Enzymic Approach to Eurythermalism of Alvinella pompejana and Its Episymbionts
Authors: CK Lee, SC Cary, AE Murray, RM Daniel
The equilibrium model, which describes the influence of temperature on enzyme activity, has been established as a valid and useful tool for characterizing enzyme eurythermalism and thermophily.
Ambion® RNAlater® Tissue Collection: RNA Stabilization Solution is an aqueous, nontoxic tissue storage reagent that rapidly permeates tissues to stabilize and protect cellular RNA.
2011
Widespread Cotranslational Formation of Protein Complexes
Authors: CDS Duncan, J Mata
Most cellular processes are conducted by multi-protein complexes. However, little is known about how these complexes are assembled. In particular, it is not known if they are formed while one or more members of the complexes are being translated (cotranslational assembly). We took a genomic approach to address this question, by systematically identifying mRNAs associated with specific proteins. In a sample of 31 proteins from Schizosaccharomyces pombe that did not contain RNA–binding domains, we found that ~38% copurify with mRNAs that encode interacting proteins. For example, the cyclin-dependent kinase Cdc2p associates with the rum1 and cdc18 mRNAs, which encode, respectively, an inhibitor of Cdc2p kinase activity and an essential regulator of DNA replication.
TRIzol® Reagent is a complete, ready-to-use reagent for the isolation of high-quality total RNA or the simultaneous isolation of RNA, DNA, and protein from a variety of biological samples.
2012
Authors: X Wang, W Zhang, F Liu et al
Our previous studies have proven the adjuvanticity of chitosan in mice when administered with inactivated and subunit influenza vaccine. In this study, we investigated the adjuvant effect of chitosan on the immunogenicity and protective efficacy of a live attenuated influenza vaccine. Mice were inoculated intranasally with live attenuated influenza vaccine plus chitosan and then challenged with a high, lethal dose of homologous or heterologous virus. Antibody responses, secretion of IFN-γ by spleen cells, body weight loss, survival rates, and residual lung virus titers were tested.
2012
Authors: D Liu, L Shi, C Han, J Yu, D Li, Y Zhang
Nicotiana benthamiana is the most widely-used experimental host in plant virology. The recent release of the draft genome sequence for N. benthamiana consolidates its role as a model for plant–pathogen interactions. Quantitative real-time PCR (qPCR) is commonly employed for quantitative gene expression analysis. For valid qPCR analysis, accurate normalisation of gene expression against an appropriate internal control is required.
2012
Authors: B-H Tzeng, Y-H Chen, C-H Huang, S-S Lin, K-R Lee, C-C Chen
Aims Restenosis is an undesirable consequence following percutaneous vascular interventions. However, the current strategy for preventing restenosis is inadequate. The aim of this study was to investigate the role of low-voltage gated T-type calcium channels in regulating vascular smooth cell (VSMC) proliferation during neointimal formation.
2012
Authors: D Kumar, P Singh, MA Yusuf, CP Upadhyaya, SD Roy, T Hohn, NB Sarin
We report the efficacy of an aldose reductase (ALDRXV4) enzyme from Xerophyta viscosa Baker in enhancing the prospects of plant’s survival under abiotic stress. Transgenic tobacco plants overexpressing ALDRXV4 cDNA showed alleviation of NaCl and mannitolinduced abiotic stress.
2012
Authors: H Xia, LL PJ Ooi, KM Hui
The down-regulation of miR-214 has previously been observed in human hepatocellular carcinoma (HCC). Here, we demonstrated the down-regulation of miR-214 is associated with cell invasion, stem-like traits and early recurrence of HCC. Firstly, we validated the suppression of miR-214 in human HCC by real-time quantitative RT-PCR (qRT-PCR) in 20 paired tumor and non-tumor liver tissues of HCC patients and 10 histologically normal liver tissues from colorectal cancer patients with liver metastases.
2012
Novel Pathways in the Pathobiology of Human Abdominal Aortic AneurysmsAuthors: I Hinterseher, R Erdman, JR Elmore, E Stahl, MC Pahl, K Derr, A Golden, JH Lillvis, MC Cindric, K Jackson, WD Bowen, CM Schworer, MA Chernousov, DP Franklin, JL Gray, RP Garvin, Z Gatalica, DJ Carey, G Tromp, H Kuivaniemi
Abdominal aortic aneurysm (AAA), a dilatation of the infrarenal aorta, typically affects males 1 65 years. The pathobiological mechanisms of human AAA are poorly understood. The goal of this study was to identify novel pathways involved in the development of AAAs. A custom-designed ‘AAA-chip’ was used to assay 43 of the differentially expressed genes identified in a previously published microarray study between AAA (n = 15) and control (n = 15) infrarenal abdominal aorta. Protein analyses were performed on selected genes.
2011
Authors: Jung-Chieh Hsua, Li-Ching Linb, Jason T.C. Tzena, Jyh-Yih Chen
The antimicrobial peptide, chrysophsin-1, exhibits antimicrobial activities with similar efficiencies for both gram-negative and gram-positive bacteria. In this study, we examined the antitumor activity and modulation of the inflammatory response of a synthetic chrysophsin-1 peptide. In vitro results showed that chrysophsin-1 had greater inhibitory effects against human fibrosarcoma (HT-1080), histiocytic lymphoma (U937), and epithelial carcinoma (HeLa) cells. LDH release by HeLa cells was comparable to that of an MTS assay after treatment with 1.5–3 μg/ml chrysophsin-1 for 24 h.
2011
Authors: RD Lin, YW Mao, SJ Leu, CY Huang, MH Lee
Many diseases occur when the immune system is weakened. Intracellular signals activate immuno-responsive cells to produce cytokines that modulate the immune response. Schisandra chinensis has been used traditionally to treat general fatigue, neurasthenia, and spontaneous sweating. In the present study, the effect of constituents of S. chinensis on cytokine release by human monocytic leukemia cells (THP-1) was tested using microparticle-based flow cytometric analysis.
2010
Characterizations of avian influenza virus H6N6 subtype isolated from domestic Muscovy duck
Authors: C Wan, G Fu, L Cheng, S Shi, H Chen, Y Huang
The full genome of A/Muscovy Duck/Fujian/FZ01/2008 (H6N6), which was first isolated from domestic Muscovy duck in Fujian Province in southern China at 2008, was cloned and sequenced by reverse transcription polymerase chain reaction (RT-PCR).
2010
Authors: RF Chen, HC Huang, CY Ou, TY Hsu, H Chuang, JC Chang, L Wang, HC Kuo, KD Yang
The prevalence of allergic diseases has increased in the past decades. It is unknown whether expression of certain microRNAs (miRNAs) in neonatal leucocytes is correlated to IgE production and/or allergic diseases. This study investigated the association of miRNA expression in neonatal leucocytes with cord blood IgE (CBIgE) elevation and development of allergic disease.
Ambion® Single Cell-to-CT™ Kit
Single cell analysis has the potential to transform life science research. The standard practice of averaging gene expression resµl ts from many cells can mask subtle, important changes occurring within single cells. To further understand the complexity of biological systems, single cell analysis must be performed.
2008
Authors: AK Ganesan, H Ho, B Bodemann, S Petersen, J Aruri, S Koshy, Z Richardson, LQ Le, T Krasieva, MG Roth, P Farmer, MA White
Melanin protects the skin and eyes from the harmful effects of UV irradiation, protects neural cells from toxic insults, and is required for sound conduction in the inner ear. Aberrant regulation of melanogenesis underlies skin disorders (melasma and vitiligo), neurologic disorders (Parkinson's disease), auditory disorders (Waardenburg's syndrome), and opthalmologic disorders (age related macular degeneration).
The Ambion® RNAqueous-Micro Kit is used for phenol-free total RNA isolation from micro-sized samples such as 10–500,000 cultured cells, as few as 10 laser capture microdissected cells, or up to 10 mg tissue using a guanidinium-based lysis⁄;denaturant and glass fiber filter separation technology.
2013
Utilization of galactooligosaccharides by Bifidobacterium longum subsp. infantis isolates
Authors: D Garrido, S Ruiz-Moyano, R Jimenez-Espinoza, H-J Eom, DE Block, DA Mills
Prebiotics are non-digestible substrates that stimulate the growth of beneficial microbial populations in the intestine, especially Bifidobacterium species. Among them, fructo- and galacto-oligosaccharides are commonly used in the food industry, especially as a supplement for infant formulas. Mechanistic details on the enrichment of bifidobacteria by these prebiotics are important to understand the effects of these dietary interventions.
2012
Authors: R Chen, H Jiang, L Li, Q Zhai, L Qi, W Zhou, X Liu, H Li, W Zheng, J Sun, C Li
Transcriptional regulation plays a central role in plant hormone signaling. At the core of transcriptional regulation is the Mediator, an evolutionarily conserved, multisubunit complex that serves as a bridge between gene-specific transcription factors and the RNA polymerase machinery to regulate transcription.
2012
Structural analysis of oval-cell–mediated liver regeneration in rats
Authors: K Dezső, V Papp, E Bugyik, H Hegyesi, G Sáfrány, C Bödör, P Nagy, S Paku
We have analyzed the architectural aspects of progenitor-cell–driven regenerative growth in rat liver by applying the 2-acetaminofluorene/partial hepatectomy experimental model. The regeneration is initiated by the proliferation of so-called oval cells. The oval cells at the proximal tips of the ductules have a more differentiated phenotype and higher proliferative rate. This preferential growth results in the formation of a seemingly random collection of small hepatocytes, called foci.
2011
Lack of Angiogenesis in Experimental Brain Metastases
Authors: E Bugyik, K Dezső, L Reiniger, V László, J Tóvári, J Tímár, P Nagy, W Klepetko, B Döme, S Paku
Angiogenesis is believed to be essential for the growth of metastatic tumors in the brain. We analyzed the vascularization of tumors formed by 4 epithelial cell lines (C38, ZR75, HT25, and H1650) and a fibrosarcoma (HT1080) cell line injected into the brains of mice. No peritumoral angiogenesis was observed. Tumors apparently acquired their vasculature by incorporation of native vessels.
2006
Authors: TJ Klisch, J Souopgui, K Juergens, B Rust, T Pieler, KA Henningfeld
We have isolated and characterized Xenopus Mxi1, a member of the Myc/Max/Mad family of bHLHZip transcription factors. Xmxi1 transcripts are present during gastrulation and early neurula stages, earlier and in broader domains as compared to the neuronal determination factor neurogenin (X-ngnr-1). Consistent with an early role in neurogenesis, Xmxi1 is positively regulated by Sox3, SoxD, and proneural genes, as well as negatively by the Notch pathway. Loss-of-function experiments demonstrate an essential role for Xmxi1 in the establishment of a mature neural state that can be activated by factors that induce neuronal differentiation, such as SoxD and X-ngnr-1. Overexpression of Xmxi1 in Xenopus embryos results in ectopic activation of Sox3, an early pan-neural marker of proliferating neural precursor cells. Within the neural plate, the neuronal differentiation marker N-tubulin and cell cycle control genes such as XPak3 and p27(Xic1) are inhibited, but the expression of early determination and differentiation markers, including X-ngnr-1 and X-MyT1, is not affected. Inhibition of neuronal differentiation by Xmxi1 is only transient, and, at early tailbud stages, both endogenous and ectopic neurogenesis are observed. While Xmxi1 enhances cell proliferation and apoptosis in the early Xenopus embryo, both activities appear not to be required for the function of Xmxi1 in primary neurogenesis.
RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE
The Ambion® RecoverAll™ Total Nucleic Acid Isolation Kit is for the extraction of total nucleic acid from formalin- or paraformalin-fixed, paraffin-embedded (FFPE) tissues. Sufficient reagents are included for 40 purifications from up to four 20 µm sections, or up to 35 mg of unsectioned core samples each.
2012
Authors: C Weng, H Dong, G Chen, Y Zhai, R Bai, H Hu, L Lu, Z Xu
Although the expression of angiogenin (ANG), an angiogenic and tumorigenic factor, is elevated in various types of cancers, its regulation mechanism remains unclear. In the present study, in silico search predicted that miR-409-3p targeted to the 3′ untranslated region (3′UTR) of the ANG mRNA. Overexpression of miR-409-3p in fibrosarcoma HT1080 cells resulted in decreased steady-state level of ANG transcript and ANG production which were achieved through direct binding of this miRNA to the ANG 3′UTR.
Ambion® ToTALLY RNA™ Total RNA Isolation Kit
The Ambion® ToTALLY RNA™ Total RNA Isolation Kit is similar to the widely used guanidine thiocyanate, acid phenol:chloroform procedure of Chomczynski and Sacchi. All reagents needed to isolate, analyze, and store RNA (other than isopropanol) are included, making the kit an excellent value. The procedure has been modified to include an optional LiCl extraction step to reduce or eliminate DNA, carbohydrate, heme, and other contaminants that can be a problem with some guanidine-based methods.
2012
Serotonin 2c receptor RNA editing in major depression and suicide
Authors: R Lyddon, AJ Dwork, M Keddache, LJ Siever, SDracheva
mRNA for serotonin 2C receptor (5-HT2CR) undergoes editing which results in numerous isoforms. More highly edited isoforms exhibit decreased function. We recently found greater 5-HT2CR editing in suicide victims with prior bipolar disorder (BPD) or schizophrenia (SZ) compared with non-suicide patients and normal controls (NC).
For the isolation of extremely high-quality RNA from yeast without the use of enzymatic pretreatments that may alter gene expression profiles. Each Ambion® kit includes sufficient reagents for 50 purifications each from 107 to 108 yeast cells.
2011
Authors: AM Porman, K Alby, MP Hirakawa, RJ Bennett
Sexual reproduction can promote genetic diversity in eukaryotes, and yet many pathogenic fungi have been labeled as obligate asexual species. It is becoming increasingly clear, however, that cryptic sexual programs may exist in some species, and that efficient mating requires the necessary developmental switch to be triggered. In this study we investigate Candida tropicalis, an important human fungal pathogen that has been reported to be asexual. Significantly, we demonstrate that C. tropicalis uses a phenotypic switch to regulate a cryptic program of sexual mating. Thus, diploid a and α cells must undergo a developmental transition to the mating- competent form, and only then does efficient cell-cell conjugation take place resulting in the formation of stable a/α tetraploids. We show that both the phenotypic switch and sexual mating depend on the conserved transcriptional regulator Wor1, which is regulated by temperature in other fungal species.
The Ambion® RiboPure™-Bacteria Kit provides a rapid, robust, column-based method for isolating high quality RNA from bacteria. Total RNA can be isolated from the toughest Gram positive bacteria samples thanks to the kit's comprehensive protocol that uses zirconia beads to thoroughly disrupt bacterial cell walls. The RiboPure™-Bacteria Kit protocol also eliminates the need for harsh treatments or enzymatic digestion which may alter gene expression profile.
2013
The Novel Cis-Encoded Small RNA h2cR Is a Negative Regulator of hfq2 in Burkholderia cenocepacia
Authors: CG Ramos, PJP da Costa, G Döring, JH Leitão
Small non-coding regulatory RNAs (sRNAs) post-transcriptionally affect multiple phenotypes in prokaryotes and eukaryotes, yet most of the underlying regulatory mechanisms and the nature of the target mRNAs remain unclear.
ChargeSwitch® Total RNA Cell kit
The ChargeSwitch® Total RNA Cell Kit is ideal for the purification of up to 25 µg of total RNA from up to 1 x 106 cultured mammalian cells or from 1 ml overnight bacterial culture, including full-length RNA and small molecular weight RNA species.
2012
Regulation of the vapBC-1 Toxin-Antitoxin Locus in Nontypeable Haemophilus influenzae
Authors: SD Cline, S Saleem, DA Daines
Nontypeable Haemophilus influenzae (NTHi) are human-adapted commensal bacteria that can cause a number of chronic mucosal infections, including otitis media and bronchitis. One way for these organisms to survive antibiotic therapy and cause recurrent disease is to stop replicating, as most antimicrobials target essential biosynthetic pathways. Toxin-antitoxin (TA) gene pairs have been shown to facilitate entry into a reversible bacteriostatic state. Characteristically, these operons encode a protein toxin and an antitoxin that associate following translation to form a nontoxic complex, which then binds to and regulates the cognate TA promoter.
siPORT™ NeoFX™ Transfection Agent
This Ambion® lipid-based formulation can be used to efficiently transfect adherent cells with siRNA as they are subcultured—without increased cytotoxicity. It is provided in one tube containing 1 mL.
2012
Dicer-Dependent Biogenesis of Small RNAs Derived from 7SL RNA
Authors: Y-F Ren, G Li, J Wu, Y-F Xue, Y-J Song, L Lv, X-J Zhang, K-F Tang
It has been reported that decreased Dicer expression leads to Alu RNAs accumulation in human retinal pigmented epithelium cells, and Dicer may process the endogenous SINE/B1 RNAs (the rodent equivalent of the primate Alu RNAs) into small interfering RNAs (siRNAs). In this study, we aimed to address whether Dicer can process Alu RNAs and their common ancestor, 7SL RNA. Using Solexa sequencing technology, we showed that Alu-derived small RNAs accounted for 0.6% of the total cellular small RNAs in HepG2.2.15 cells, and the abundance decreased when Dicer was knocked down.
2012
Novel molecular beacons to monitor microRNAs in non-small-cell lung cancer
Authors: Q Yao, A-m Zhang, H Ma, S Lin, X-x Wang, J-g Sun, Z-t Chen
Lung cancer is the leading cause of cancer death worldwide. There is no effective early diagnostic technology for lung cancer. microRNAs (miRNAs) are noncoding RNA molecules which regulate the process of cell growth and differentiation in human cancers. Hsa-miR-155 (miR-155), highly expressed in non-small-cell lung cancer (NSCLC), can be used as a diagnostic marker for NSCLC.
2011
Authors: N Zhu, D Zhang, S Chen, X Liu, L Lin, X Huang, Z Guo, J Liu, Y Wang, W Yuan, Y Qin
Inflammation is observed at all stages of atherosclerosis. The initial stage of atherosclerosis is characterized by recruitment of leukocytes to activated endothelial cells (ECs). MicroRNAs (miRNAs) are a class of 19–25 nucleotides, non-protein-coding RNAs that repress target gene expression by translational inhibition or mRNA degradation.
2008
Authors: Y Wang, ATC Lee, JZI Ma, J Wang, J Ren, Y Yang, E Tantoso, K-B Li, LLPJ Ooi, P Tan, CGL Lee
Like other cancers, aberrant gene regulation features significantly in hepatocellular carcinoma (HCC). MicroRNAs (miRNAs) were recently found to regulate gene expression at the post-transcriptional/translational levels. The expression profiles of 157 miRNAs were examined in 19 HCC patients, and 19 up-regulated and 3 down-regulated miRNAs were found to be associated with HCC. Putative gene targets of these 22 miRNAs were predicted in silico and were significantly enriched in 34 biological pathways, most of which are frequently dysregulated during carcinogenesis.M-MLV (Moloney Murine Leukemia Virus) Reverse Transcriptase (RT) is a recombinant DNA polymerase that synthesizes a complementary DNA strand from single-stranded RNA, DNA, or an RNA:DNA hybrid.
2012
Authors: Y Li, Y Zhang, R Li, W Chen, M Howell, R Zhang, G Chen
The roles of vitamin A (VA) in the development of metabolic diseases remain unanswered. We have reported that retinoids synergized with insulin to induce the expression of sterol-regulatory element-binding protein 1c gene (Srebp-1c) expression in primary rat hepatocytes. Additionally, the hepatic Srebp-1c expression is elevated in Zucker fatty (ZF) rats, and reduced in those fed a VA deficient diet. VA is metabolized to retinoic acid (RA) for regulating gene expression.
2008
Authors: JM Gonzalez, RD Dijkhuis, DD Johnson, JN Carter, SE Johnson
Ractopamine-HCl (RAC) is a beta-adrenergic agonist with variable effects on cattle performance and carcass variables. Cull cows fed RAC (200 mg . head(-1) . d(-1)) demonstrate an increased size of type I and II muscle fibers that does not translate into a larger ribeye area. The objective of this study was to examine the dose-dependent effects of RAC on cull cow muscle morphometrics. Eighty-eight cull beef cows representing 2 breed types (n = 44 each) were fed 0, 100, 200, and 300 mg . head(-1) . d(-1) of RAC for the last 28 d of a 54-d feeding period. On d 54, cows were slaughtered, and samples of the LM and semimembranosus muscle (SM) from 16 randomly selected carcasses (n = 4 per treatment) were taken for measurement of beta (2)-adrenergic receptors and type I, IIA, and IIX myosin heavy chain (MyHC) gene expression.
For the reverse transcription of RNA. The Ambion® kit includes a M-MLV RT that maintains full RNase H activity for increased sensitivity of detection in RT-PCR experiments. Includes sufficient reagents for 50 reactions.
2012
Stable changes in CD4+ T lymphocyte miRNA expression after exposure to HIV-1
Authors: F Bignami, E Pilotti, L Bertoncelli, P Ronzi, M Gulli, N Marmiroli, G Magnani, M Pinti, L Lopalco, C Mussini, R Ruotolo, M Galli, A Cossarizza, C Casoli
MicroRNAs (miRNAs) inhibit HIV-1 expression by either modulating host innate immunity or by directly interfering with viral mRNAs. We evaluated the expression of 377 miRNAs in CD4+ T cells from HIV-1 élite long-term nonprogressors (éLTNPs), naive patients, and multiply exposed uninfected (MEU) patients, and we observed that the éLTNP patients clustered with naive patients, whereas all MEU subjects grouped together. The discriminatory power of miRNAs showed that 21 miRNAs significantly differentiated éLTNP from MEU patients and 23 miRNAs distinguished naive from MEU patients, whereas only 1 miRNA (miR-155) discriminated éLTNP from naive patients.
The Ambion® RETROscript® First Strand Synthesis Kit includes reagents for reverse transcription of RNA into double-stranded cDNA products. Includes sufficient reagents for 40 reactions.
2012
Authors: MA Siddiqui, K Malathi
Autophagy is a tightly regulated mechanism that mediates sequestration, degradation, and recycling of cellular proteins, organelles, and pathogens. Several proteins associated with autophagy regulate host responses to viral infections.
High Capacity cDNA Reverse Transcription Kit
The High Capacity cDNA Reverse Transcription Kit (formerly the High Capacity cDNA Archive Kit) delivers extremely high-quality, single-stranded cDNA from 0.02 to 2 µg input total RNA. Ideal for generating cDNA archives. The High Capacity cDNA Reverse Transcription Kit can convert up to 2 µg of input total RNA in a single 20 µl reaction. Reactions can be scaled up to 100 µl to generate 10 µg of cDNA from a single reaction The cDNA generated is ideal for a range of applications including real-time PCR, standard PCR, and microarrays.
2012
Authors: Y He, Y Yu, Y Zhang, J Song, A Mitra, Y Zhang, Y Wang, D Sun, S Zhang
Gene expression of lymphocytes was found to be influenced by histone methylation in mammals and trimethylation of lysine 27 on histone H3 (H3K27me3) normally represses genes expressions. Peripheral blood lymphocytes are the main source of somatic cells in the milk of dairy cows that vary frequently in response to the infection or injury of mammary gland and number of parities.
2012
Trim28 Is Required for Epigenetic Stability During Mouse Oocyte to Embryo Transition
Authors: DM Messerschmidt, W de Vries, M Ito, D Solter, A Ferguson-Smith, BB Knowles
Phenotypic variability in genetic disease is usually attributed to genetic background variation or environmental influence. Here, we show that deletion of a single gene, Trim28 (Kap1 or Tif1β), from the maternal germ line alone, on an otherwise identical genetic background, results in severe phenotypic and epigenetic variability that leads to embryonic lethality. We identify early and minute epigenetic variations in blastomeres of the preimplantation embryo of these animals, suggesting that the embryonic lethality may result from the misregulation of genomic imprinting in mice lacking maternal Trim28. Our results reveal the long-range effects of a maternal gene deletion on epigenetic memory and illustrate the delicate equilibrium of maternal and zygotic factors during nuclear reprogramming.
Novel transcription reaction conditions and Ambion's patented, high-yield technology allow the synthesis of 10–50 times the amount of RNA produced by conventional transcription reactions. Each Ambion® kit includes sufficient reagents for 200 reactions.
2012
Authors: Zhi-Rui Liu, Jie Tao, Bang-Qian Dong, Gang Ding, Zhi-Jun Cheng, Hui-Qiong He, Yong-Hua Ji
Chitin synthase (CHS) is an enzyme that is required for chitin formation in insect cuticles and other tissues. In this study, CHS genes from two destructive rice insect pests, the brown planthopper Nilaparvata lugens and the small brown planthopper Laodelphax striatellus, were cloned. Phylogenetic analysis showed that these genes belonged to class CHS1 of the CHS gene family.
2012
Authors: IE Catrina, SAE Marras, DP Bratu
New approaches for imaging dynamic processes involving RNAs in living cells are continuously being developed and optimized. The use of molecular beacons synthesized from 2′-O-methylribonucleotides (which are resistant to cellular nucleases) is an established approach for visualizing native mRNAs in real time.
2012
Authors: Y Liu, K Timani, X Ou, HE Broxmeyer, JJ He
We reported previously that HIV-1 the Tat-interacting protein of 110 kDa (TIP110; P110(NRB)/SART3/p110) is important in regulation of hematopoiesis, and in maintaining pluripotent factor (NANOG, OCT4, and SOX2) expression in and pluripotency of human embryonic stem cells (hESCs). Here we show that TIP110 expression in hESCs line H9 and embryonal carcinoma cell line NT-2 is regulated by C-MYC expression in ESCs through an E box present in the TIP110 promoter region. Through up- and down- modulation of expression, TIP110 induces OCT4 exon 1a inclusion and exon 1b skipping in our OCT4 minigene model. Thus, TIP110 expression in ESCs regulates alternative splicing of OCT4, an event required for pluripotency of ESCs.
MessageAmp™ II aRNA Amplification Kit
The Ambion® MessageAmp™ II aRNA Amplification Kit is for the amplification of as little as 100 pg of input RNA for GeneChip® analysis. The kit includes sufficient reagents for 20 aRNA amplifications.
2011
Authors: D Li, P Chen, X-Y Li, L-Y Zhang, W Xiong, M Zhou, L Xiao, F Zeng, X-L Li, M-H Wu, G-Y Li
Although several miRNAs have been identified to be involved in glioblastoma tumorigenesis, little is known about the global expression profiles of miRNAs and their functional targets in astrocytomas at earlier stages of malignancy. In this study the global expression of miRNAs and mRNAs in normal brain tissue samples and grade I–III astrocytomas were analyzed parallelly using microarrays, and the grade-specific expression profiles of them were obtained by unsupervised hierarchical clustering.
2012
Authors: IE Catrina, SAE Marras, DP Bratu
New approaches for imaging dynamic processes involving RNAs in living cells are continuously being developed and optimized. The use of molecular beacons synthesized from 2′-O-methylribonucleotides (which are resistant to cellular nucleases) is an established approach for visualizing native mRNAs in real time.
2012
Authors: Y Liu, K Timani, X Ou, HE Broxmeyer, JJ He
We reported previously that HIV-1 the Tat-interacting protein of 110 kDa (TIP110; P110(NRB)/SART3/p110) is important in regulation of hematopoiesis, and in maintaining pluripotent factor (NANOG, OCT4, and SOX2) expression in and pluripotency of human embryonic stem cells (hESCs). Here we show that TIP110 expression in hESCs line H9 and embryonal carcinoma cell line NT-2 is regulated by C-MYC expression in ESCs through an E box present in the TIP110 promoter region. Through up- and down- modulation of expression, TIP110 induces OCT4 exon 1a inclusion and exon 1b skipping in our OCT4 minigene model. Thus, TIP110 expression in ESCs regulates alternative splicing of OCT4, an event required for pluripotency of ESCs.
Anti-miR™ hsa-let-7c miRNA Inhibitor Postive Control
Ambion® Anti-miR™ miRNA Inhibitors are chemically modified, single-stranded nucleic acids designed to specifically bind to and inhibit endogenous microRNA (miRNA) molecules.
2012
Authors: N Nadiminty, R Tummala, W Lou, Y Zhu, J Zhang, X Chen, RW eVere White, HJ Kung, CP Evans, AC Gao
Castration-resistant prostate cancer continues to rely on androgen receptor (AR) expression. AR plays a central role in the development of prostate cancer and progression to castration resistance during and after androgen deprivation therapy. Here, we identified miR-let-7c as a key regulator of expression of AR. miR-let-7c suppresses AR expression and activity in human prostate cancer cells by targeting its transcription via c-Myc. Suppression of AR by let-7c leads to decreased cell proliferation of human prostate cancer cells. Down-regulation of Let-7c in prostate cancer specimens is inversely correlated with AR expression, whereas the expression of Lin28 (a repressor of let-7) is correlated positively with AR expression. Our study demonstrates that the miRNA let-7c plays an important role in the regulation of androgen signaling in prostate cancer by down-regulating AR expression. These results suggest that reconstitution of miR-let-7c may aid in targeting enhanced and hypersensitive AR in advanced prostate cancer.
Anti-miR™ miRNA Inhibitor Negative Control #1
Ambion® Anti-miR™ miRNA Inhibitors are chemically modified, single-stranded nucleic acids designed to specifically bind to and inhibit endogenous microRNA (miRNA) molecules.
2012
Authors: Castro RE, Ferreira DM, Afonso MB, Borralho PM, Machado MV, Cortez-Pinto H, Rodrigues CM.
BACKGROUND & AIMS:
Non-alcoholic fatty liver disease (NAFLD) comprises a spectrum of stages from simple steatosis to non-alcoholic steatohepatitis (NASH). However, disease pathogenesis remains largely unknown. microRNA (miRNA or miR) expression has recently been reported to be altered in human NASH, and modulated by ursodeoxycholic acid (UDCA) in the rat liver. Here, we aimed at evaluating the miR-34a/Sirtuin 1(SIRT1)/p53 pro-apoptotic pathway in human NAFLD, and to elucidate its function and modulation by UDCA in the rat liver and primary rat hepatocytes.
Ambion® Pre-miR™ miRNA Precursor Molecules are small, chemically modified, double-stranded RNA molecules designed to mimic endogenous mature miRNAs. This ready-to-use miRNA mimic can be introduced into cells using transfection or electroporation parameters similar to those used for siRNAs.
2012
Authors: T-C Huang, H-Y Chang, C-Y Chen, P-Y Wu, H Lee, Y-F Liao, W-M Hsu, H-C Huang, H-F Juan
Neuroblastoma is the most common extracranial solid tumor in children. We investigate whether miR-124, the abundant neuronal miRNA, plays a pivotal role in neuroblastoma. Knockdown of miR-124 promotes neuroblastoma SK-N-SH cell differentiation, cell cycle arrest and apoptosis. Further miR-124 is predicted to target aryl hydrocarbon receptor (AHR) which may promote neuroblastoma cell differentiation. We validate that miR-124 may suppress the expression of AHR by targeting its 3′-UTR. These results suggest that miR-124 could serve as a potential therapeutic target of neuroblastoma.
2011
TNF-α is a novel target of miR-19a
Authors: M Liu, Z Wang, S Yang, W Zhang, S He, C Hu, H Zhu, L Quan, J Bai1, N Xu
Many studies have demonstrated the overexpression and amplification of the miR-17-92 cluster in malignant human cancers, including B-cell lymphomas and lung cancers. The purpose of this study was to investigate for the first time, the expression of the miR-17-92 cluster in esophageal squamous cell carcinoma (ESCC). The miR-17-92 cluster was found to be overexpressed in 21 out of 28 (75%) esophageal cancer samples.
2011
Authors: MR Epis, A Barker, KM Giles, DJ Beveridge, PJ Leedman
ERBB-2 overexpression is associated with the development and progression of cancer and mediates its resistance to therapy. It has been suggested that post-transcriptional mechanisms control the overexpression of ERBB-2 in prostate cancer (PCa).
2010
Prox1 expression is negatively regulated by miR-181 in endothelial cells
Authors: J Kazenwadel, MZ Michael, NL Harvey
The specification of arterial, venous, and lymphatic endothelial cell fate is critical during vascular development. Although the homeobox transcription factor, Prox1, is crucial for the specification and maintenance of lymphatic endothelial cell identity, little is known regarding the mechanisms that regulate Prox1 expression.
miRNA mimics are small, chemically modified double-stranded RNAs that mimic endogenous miRNAs and enable miRNA functional analysis by up-regulation of miRNA activity.
2012
Authors: H-H Kwok, P Y-K Yue, N-K Mak, R N-S Wong
Wrinkle formation is one of the primary characteristics of skin aging, the major cause of wrinkle is the loss of structural protein type I collagen in dermal layer of skin. Topical application of natural substances to reduce wrinkle is gaining attention in recent years. Although a number of polyphenoic compounds are suggested to prevent ultraviolet-induced wrinkle, very few of them are able to increase type I collagen synthesis directly. Ginseng has been known in folk medicine of its beneficial effect to skin.
mirVana™ miRNA Probe Construction Kit
The Ambion® mirVana™ miRNA Probe Construction Kit uses in vitro transcription to generate short (<100 nt) radiolabeled or nonisotopically labeled RNA probes in less than 1 hour. The mirVana™ miRNA Probe Construction Kit includes reagents for preparing 30 in vitro transcription templates as well as reagents for 30 in vitro transcription reactions.
2013
MicroRNAs 221 and 222 regulate the undifferentiated state in mammalian male germ cells
Authors: QE Yang, KE Racicot, AV Kaucher, MJ Oatley, JM Oatley
Continuity of cycling cell lineages relies on the activities of undifferentiated stem cell-containing subpopulations. Transition to a differentiating state must occur periodically in a fraction of the population to supply mature cells, coincident with maintenance of the undifferentiated state in others to sustain a foundational stem cell pool. At present, molecular mechanisms regulating these activities are poorly defined for most cell lineages. Spermatogenesis is a model process that is supported by an undifferentiated spermatogonial population and transition to a differentiating state involves attained expression of the KIT receptor.
Ambion® In Vivo Negative Control #1 siRNA
Ambion® In Vivo siRNAs are designed using the proven Silencer® Select algorithm and incorporate chemical modifications for superior serum stability with in vivo delivery. The added serum stability does not compromise the performance of the siRNAs. Ambion® In Vivo siRNAs have been shown to be non-toxic in vivo (mouse) and non-immunogenic (cell-based assays). In cell-based assays, Ambion® In Vivo siRNAs exhibit equivalent or better potency than Silencer
2010
Single-Nucleotide Polymorphisms Inside MicroRNA Target Sites Influence Tumor Susceptibility
Authors: MS Nicoloso, H Sun, R Spizzo, H Kim, P Wickramasinghe, M Shimizu, SE Wojcik, J Ferdin, T Kunej, L Xiao, S Manoukian, G Secreto, F Ravagnani, X Wang, P Radice, CM Croce, RV Davuluri, GA Calin
Single-nucleotide polymorphisms (SNP) associated with polygenetic disorders, such as breast cancer (BC), can create, destroy, or modify microRNA (miRNA) binding sites; however, the extent to which SNPs interfere with miRNA gene regulation and affect cancer susceptibility remains largely unknown. We hypothesize that disruption of miRNA target binding by SNPs is a widespread mechanism relevant to cancer susceptibility.
MICROBExpress™ Bacterial mRNA Enrichment Kit
This Ambion® kit is for the purification of bacterial mRNA by removing rRNA from total RNA.
Authors: Q Leroy, K Lebrigand, F Armougom, P Barbry, R Thiéry, D Raoult
Coxiella burnetii, the causative agent of the zoonotic disease Q fever, is mainly transmitted to humans through an aerosol route. A spore-like form allows C. burnetii to resist different environmental conditions. Because of this, analysis of the survival strategies used by this bacterium to adapt to new environmental conditions is critical for our understanding of C. burnetii pathogenicity. Here, we report the early transcriptional response of C. burnetii under temperature stresses. Our data show that C. burnetii exhibited minor changes in gene regulation under short exposure to heat or cold shock. While small differences were observed, C. burnetii seemed to respond similarly to cold and heat shock. The expression profiles obtained using microarrays produced in-house were confirmed by quantitative RT-PCR.
Ambion® TURBO DNA-free™ contains reagents for the efficient, complete digestion of DNA along with the removal of the enzyme and divalent cations post-digestion.
2012
Safety and Efficacy of Topical Infliximab in a Mouse Model of Ocular Surface Scarring
Authors: G Ferrari, F Bignami, C Giacomini, S Franchini, P Rama
2012
Authors: Q Zeng, MD Laiosa, DA Steeber, EM Biddle, Q Peng, C-H Yang
Dickeya dadantii 3937 is a gram-negative phytopathogenic bacterium that expresses genes encoding a type III secretion system (T3SS) in a bistable pattern when cultured in a homogeneous minimal media. In this work, we further characterized the bistable gene expression of T3SS at the single-cell level.
2012
Authors: TJ Stark, JD Arnold, DH Spector, GW Yeo
Human cytomegalovirus (HCMV) contributes its own set of microRNAs (miRNAs) during lytic infection of cells, likely fine-tuning conditions important for viral replication. To enhance our understanding of this component of the HCMV-host transcriptome, we have conducted deep-sequencing analysis of small RNAs (smRNA-seq) from infected human fibroblast cells. We found that HCMV-encoded miRNAs accumulate to ∼20% of the total smRNA population at late stages of infection, and our analysis led to improvements in viral miRNA annotations and identification of two novel HCMV miRNAs, miR-US22 and miR-US33as.
2012
Characterization of Microbial Population Shifts during Sample Storage
Authors: HJ Mills, BK Reese, C St. Peter
The objective of this study was to determine shifts in the microbial community structure and potential function based on standard Integrated Ocean Drilling Program (IODP) storage procedures for sediment cores. Standard long-term storage protocols maintain sediment temperature at 4°C for mineralogy, geochemical, and/or geotechnical analysis whereas standard microbiological sampling immediately preserves sediments at −80°C. Storage at 4°C does not take into account populations may remain active over geologic time scales at temperatures similar to storage conditions.
2012
PpsR, a Regulator of Heme and Bacteriochlorophyll Biosynthesis, Is a Heme-sensing Protein
Authors: L Yin, V Dragnea, CE Bauer
Heme-mediated regulation, presented in many biological processes, is achieved in part with proteins containing heme regulatory motif. In this study, we demonstrate that FLAG-tagged PpsR isolated from Rhodobacter sphaeroides cells contains bound heme.
2012
Authors: PE Griffin, LF Roddam, YC Belessis, R Strachan, S Beggs, A Jaffe, MA Cooley
The Pseudomonas aeruginosa quorum sensing signal molecule N-3-oxododecanoyl-L-homoserine lactone (3OC12HSL) can inhibit function of the mammalian anti-inflammatory transcription factor peroxisome proliferator activated receptor (PPAR)γ, and can be degraded by human paraoxonase (PON)2. Because 3OC12HSL is detected in lungs of cystic fibrosis (CF) patients infected with P. aeruginosa, we investigated the relationship between P. aeruginosa infection and gene expression of PPARγ and PON2 in bronchoalveolar lavage fluid (BALF) of children with CF.
2012
RNA-Seq and molecular docking reveal multi-level pesticide resistance in the bed bug
Authors: P Mamidala, AJ Wijeratne, S Wijeratne, K Kornacker, B Sudhamalla, LJ Rivera-Vega, A Hoelmer, T Meulia, SC Jones, O Mittapalli
Bed bugs (Cimex lectularius) are hematophagous nocturnal parasites of humans that have attained high impact status due to their worldwide resurgence. The sudden and rampant resurgence of C. lectularius has been attributed to numerous factors including frequent international travel, narrower pest management practices, and insecticide resistance.
2011
Protocol: A simple phenol-based method for 96-well extraction of high quality RNA from Arabidopsis
Authors: MS Box, V Coustham, C Dean, JS Mylne
Many experiments in modern plant molecular biology require the processing of large numbers of samples for a variety of applications from mutant screens to the analysis of natural variants. A severe bottleneck to many such analyses is the acquisition of good yields of high quality RNA suitable for use in sensitive downstream applications such as real time quantitative reverse-transcription-polymerase chain reaction (real time qRT-PCR). Although several commercial kits are available for high-throughput RNA extraction in 96-well format, only one non-kit method has been described in the literature using the commercial reagent TRIZOL.
2011
Genomic Characterization and High Prevalence of Bocaviruses in Swine
Authors: T Shan, D Lan, L Li, C Wang, L Cui, W Zhang, X Hua, C Zhu, W Zhao, E Delwart
Using random PCR amplification followed by plasmid subcloning and DNA sequencing, we detected bocavirus related sequences in 9 out of 17 porcine stool samples. Using primer walking, we sequenced the nearly complete genomes of two highly divergent bocaviruses we provisionally named porcine bocavirus 1 isolate H18 (PBoV1-H18) and porcine bocavirus 2 isolate A6 (PBoV2-A6) which differed by 51.8% in their NS1 protein.
2011
Authors: S Shuxian, W Sheng, S Schachtele, J Lokensgard
Production of reactive oxygen species (ROS) and proinflammatory cytokines by microglial cells in response to viral brain infection contributes to both pathogen clearance and neuronal damage. In the present study, we examined the effect of herpes simplex virus (HSV)-1-induced, NADPH oxidase-derived ROS in activating mitogen-activated protein kinases (MAPKs) as well as driving cytokine and chemokine expression in primary murine microglia.
2011
Authors: M Panigaj, H Glier, M Wildova, K Holada
Prion diseases have been observed to deregulate the transcription of erythroid genes, and prion protein knockout mice have demonstrated a diminished response to experimental anemia. To investigate the role of the cellular prion protein (PrPC) in erythropoiesis, we studied the protein’s expression on mouse erythroid precursors in vivo and utilized an in vitro model of the erythroid differentiation of murine erythroleukemia cells (MEL) to evaluate the effect of silencing PrPC through RNA interference. The expression of PrPC and selected differentiation markers was analyzed by quantitative multicolor flow cytometry, western blot analysis and quantitative RT-PCR. The silencing of PrPC expression in MEL cells was achieved by expression of shRNAmir from an integrated retroviral vector genome.
2009
Krüppel-like factor 4 regulates adaptive expression of the zinc transporter Zip4 in mouse small intestine
Authors: JP Liuzzi, L Guo, S-M Chang, RJ Cousins
Epithelial cells of the small intestine are the site of zinc absorption. Intestinal uptake of zinc is inversely proportional to the dietary supply of this essential micronutrient. The mechanism responsible for this adaptive differential in apical zinc transport is not known. The zinc transporter Zip4 (Slc39a4) is essential for adequate enteric zinc uptake.
Acid Phenol:Chloroform:IAA (125:24:1) is premixed and supplied at pH 4.5 ± 0.2. Provided in one bottle of 100 mL. In RNA extraction procedures, Acid Phenol:Chloroform:IAA aids in the removal of DNA (it partitions into the organic phase), helps to stabilize the interface, and prevents foaming when mixing.
2008
Authors: S Ramanan, M Kooshkib, W Zhaob, F-C Hsud, ME Robbins
Whole-brain irradiation (WBI) can lead to cognitive impairment several months to years after irradiation. Studies on rodents have shown a rapid and sustained increase in activated microglia (brain macrophages) following brain irradiation, contributing to a chronic inflammatory response and a corresponding decrease in hippocampal neurogenesis. Thus, alleviating microglial activation following radiation represents a key strategy to minimize WBI-induced morbidity. We hypothesized that pretreatment with peroxisomal proliferator-activated receptor (PPAR)α agonists would ameliorate the proinflammatory responses seen in the microglia following in vitro radiation. Irradiating BV-2 cells (a murine microglial cell line) with single doses (2–10 Gy) of 137Cs γ-rays led to increases in (1) the gene expression of IL-1β and TNFα, (2) Cox-2 protein levels, and (3) intracellular ROS generation.
For the polyadenylation of in vitro transcribed RNA to enhance translation initiation efficiency. Sufficient reagents are included for 25 reactions.
2012
Expression profile of miRNAs in Populus cathayana L. and Salix matsudana Koidz under salt stress
Authors: J Zhou, M Liu, J Jiang, G Qiao, S Lin, H Li, L Xie, R Zhuo
Soil salinization can lead to environmental and ecological problems worldwide. Abiotic stressors, including salinity, are suspected to regulate microRNA (miRNA) expression. Plants exposed to such abiotic stressors express specific miRNAs, which are genes encoding small non-coding RNAs of 20–24 nucleotides. miRNAs are known to exist widely in plant genomes, and are endogenous.
2011
Label-Free MicroRNA Profiling Not Biased by 3' End 2'-O-Methylation
Authors: Y Shen, K-x Zheng, D Duan, L Jiang, J Li
Accurate quantification of miRNA expression level is essential to the study of its biology, and many cutting-edge technologies have been developed to accommodate this need. Yet most of them were designed primarily for the “regular” RNAs such as animal miRNAs and may overlook the fact that plant miRNAs and many other small noncoding RNAs are 2′-O-methylated at the 3′ end nucleotide. According to our experimental data and previous reports, this structural variation is detrimental to the effectiveness of the commonly used enzymatic labeling methods, leading to strongly biased results (24-fold difference).
The Ambion® MEGAclear™ Kit is designed for the quick cleanup of any large-scale transcription reaction. MEGAclear™ efficiently separates RNA from unincorporated NTPs, enzymes, and buffer components. Each kit includes sufficient reagents for 20 reactions.
2012
Highly Efficient and Specific Genome Editing in Silkworm Using Custom TALENs
Authors: S Ma, S Zhang, F Wang, Y Liu, Y Liu, H Xu, C Liu, Y Lin, P Zhao, Q Xia
Establishment of efficient genome editing tools is essential for fundamental research, genetic engineering, and gene therapy. Successful construction and application of transcription activator-like effector nucleases (TALENs) in several organisms herald an exciting new era for genome editing. We describe the production of two active TALENs and their successful application in the targeted mutagenesis of silkworm, Bombyx mori, whose genetic manipulation methods are parallel to those of Drosophila and other insects.
Ambion®'s RNase-free DNase I is of the highest purity available and is recommended to degrade DNA in the presence of RNA when the absence of RNase is critical to maintain the integrity of the RNA.
2012
Authors: TI Jones, JCJ Chen, F Rahimov, S Homma, P Arashiro, ML Beermann, OD King, JB Miller, LM Kunkel, CP Emerson Jr, KR Wagner, PL Jones
Facioscapulohumeral muscular dystrophy (FSHD), the most prevalent myopathy afflicting both children and adults, is predominantly associated with contractions in the 4q35-localized macrosatellite D4Z4 repeat array.
2012
Authors: LS Reinert, L Harder, CK Holm, MB Iversen, KA Horan, F Dagnæs-Hansen, BP Ulhøi, TH Holm, TH Mogensen, T Owens, JR Nyengaard, AR Thomsen, SR Paludan
Herpes simplex viruses (HSVs) are highly prevalent neurotropic viruses. While they can replicate lytically in cells of the epithelial lineage, causing lesions on mucocutaneous surfaces, HSVs also establish latent infections in neurons, which act as reservoirs of virus for subsequent reactivation events. Immunological control of HSV involves activation of innate immune pattern-recognition receptors such as TLR3, which detects doublestranded RNA and induces type I IFN expression.
2012
Validation of Reference Genes for Gene Expression Studies in Aphis glycines (Hemiptera: Aphididae)
Authors: R Bansal, P Mamidala, MA Rouf Mian, O Mittapalli, AP Michel
Quantitative real-time polymerase chain reaction (qRT-PCR) is a common and robust tool for accurate quantification of mRNA transcripts. To normalize results, a housekeeping gene ([HKG], reference gene or endogenous control gene) is mandatory. Soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae), is a significant soybean, Glycine max (L.) Merr., pest, yet gene expression and functional genomics studies are hindered by a lack of stable HKGs.
2012
Epigenetic silencing of myogenic gene program by Myb-binding protein 1a suppresses myogenesis
Authors: C-C Yang, H Liu, SL Chen, T-H Wang, C-L Hsieh, Y Huang, S-J Chen, H-Cn Chen, B Yat-Ming Yung, B Chin-Ming Tan
Skeletal myogenesis involves highly coordinated steps that integrate developmental cues at the chromatin of muscle progenitors. Here, we identify Myb-binding protein 1a (Mybbp1a) as a novel negative regulator of muscle-specific gene expression and myoblast differentiation. The mode of action of Mybbp1a was linked to promoter regulation as illustrated by its interaction with MyoD at the genomic regions of silent muscle-specific genes as well as its negative effect on MyoD-mediated transcriptional activity. We propose that Mybbp1a exerts its repressive role by inducing a less permissible chromatin structure following recruitment of negative epigenetic modifiers such as HDAC1/2 and Suv39h1.
2011
Wnt Signaling Mediates Pathological Vascular Growtin h Proliferative Retinopathy
Authors: J Chen, A Stahl, NM Krah, MR Seaward, RJ Dennison, P Sapieha, J Hua, CJ Hatton, AM Juan, CM Aderman, KL Willett, KI Guerin, A Mammoto, M Campbell, LEH Smith
Ischemic proliferative retinopathy, characterized by pathological retinal neovascularization, is a major cause of blindness in working-age adults and children. Defining the molecular pathways distinguishing pathological neovascularization from normal vessels is critical to controlling these blinding diseases with targeted therapy. Because mutations in Wnt signaling cause defective retinal vasculature in humans with some characteristics of the pathological vessels in retinopathy, we investigated the potential role of Wnt signaling in pathological retinal vascular growth in proliferative retinopathy.
2004
Organ-Specific Silencing of Duplicated Genes in a Newly Synthesized Cotton Allotetraploid
Authors: KL Adams, R Percifield, JF Wendel
Most eukaryotes have undergone genome doubling at least once during their evolutionary history. Hybridization followed by genome doubling (allopolyploidization) is a prominent mode of speciation in plants, leading to phenotypic novelty and changes in genome structure and gene expression. Molecular events that take place immediately after polyploid formation can be studied using newly synthesized allopolyploids. Here we studied the extent of gene silencing in a newly created and genomically stable allotetraploid cotton, of genotype AAGG, using an AFLP-cDNA display screen. Over 2000 transcripts were screened and ~ 5% of the duplicated genes in the allotetraploid were inferred to have been silenced or downregulated. Sequencing of 24 AFLP-cDNA fragments revealed genes with a variety of functions
Used as a component in many hybridization protocols, Ambion® Salmon Sperm DNA is rigorously tested for RNase and DNase contamination and is suspended in nuclease-free water at a concentration of 10 mg⁄mL.
2011
Authors: Ravindra B. Chalamalasetty, William C. Dunty Jr, Kristin K. Biris,Rieko Ajima, Michelina Iacovino, Arica Beisaw, Lionel Feigenbaum, Deborah L. Chapman, Jeong Kyo Yoon, Michael Kyba & Terry P. Yamaguchi
Segmentation is an organizing principle of body plans. The segmentation clock, a molecular oscillator best illustrated by the cyclic expression of Notch signalling genes, controls the periodic cleavage of somites from unsegmented presomitic mesoderm during vertebrate segmentation. Wnt3a controls the spatiotemporal expression of cyclic Notch genes; however, the underlying mechanisms remain obscure. Here we show by transcriptional profi ling of Wnt3a−/− embryos that the bHLH transcription factor, Mesogenin1 (Msgn1) , is a direct target gene of Wnt3a. To identify Msgn1 targets, we conducted genome-wide studies of Msgn1 activity in embryonic stem cells. We show that Msgn1 is a major transcriptional activator of a Notch signalling program and synergizes with Notch to trigger clock gene expression. Msgn1 also indirectly regulates cyclic genes in the Fgf and Wnt pathways. Thus, Msgn1 is a central component of a transcriptional cascade that translates a spatial Wnt3a gradient into a temporal pattern of clock gene expression.
Ambion® ULTRAhyb®-Oligo is specifically formulated for use with oligonucleotide probes in northern analysis. It contains a unique blend of hybridization accelerators and blocking agents that greatly enhance the levels of hybridization so that signals that once took days to visualize are now apparent in hours. It is supplied in one bottle containing 125 mL.
2008
Authors: AK Ebralidze, FC Guibal, U Steidl, P Zhang, S Lee, B Bartholdy, MA Jorda, V Petkova, F Rosenbauer, G Huang, T Dayaram, J Klupp, KB O’Brien, B Will, M Hoogenkamp, KLB Borden, C Bonifer, DG Tenen
The transcription factor PU.1 is an important regulator of hematopoiesis; precise expression levels are critical for normal hematopoietic development and suppression of leukemia.
The Ambion® GLOBINclear™-Mouse⁄Rat Kit employs a novel, non-enzymatic technology that rapidly depletes >95% of the alpha and beta globin mRNA from total RNA preparations derived from whole blood. The kit includes sufficient reagents for 20 rxns.
2012
Novel roles for KLF1 in erythropoiesis revealed by mRNA-seq
Authors: MR Tallack, GW Magor, B Dartigues, et al.
KLF1 (formerly known as EKLF) regulates the development of erythroid cells from bi-potent progenitor cells via the transcriptional activation of a diverse set of genes. Mice lacking Klf1 die in utero prior to E15 from severe anemia due to the inadequate expression of genes controlling hemoglobin production, cell membrane and cytoskeletal integrity, and the cell cycle. We have recently described the full repertoire of KLF1 binding sites in vivo by performing KLF1 ChIP-seq in primary erythroid tissue (E14.5 fetal liver). Here we describe the KLF1-dependent erythroid transcriptome by comparing mRNA-seq from Klf1+/+ and Klf1-/-erythroid tissue. This has revealed novel target genes not previously obtainable by traditional microarray technology and provided novel insights into the function of KLF1 as a transcriptional activator. We define a cis regulatory module bound by KLF1, GATA1, TAL1 and Ep300 that co-ordinates a core set of erythroid genes. We also describe a novel set of erythroid specific promoters that drive high level expression of otherwise ubiquitously expressed genes in erythroid cells. Our study has identified two novel lncRNAs that are dynamically expressed during erythroid differentiation and discovered a role for KLF1 in directing apoptotic gene expression to drive the terminal stages of erythroid maturation.
Silencer® siRNA Construction Kit
The Ambion® Silencer® siRNA Construction Kit is for the synthesis of siRNAs by in vitro transcription of DNA templates and costs less per siRNA than chemical synthesis. The kit includes sufficient reagents to synthesize 15 siRNAs.
2011
N6-Methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO
Authors: G Jia, Y Fu, X Zhao, Q Dai, G Zheng, Y Yang, C Yi, T Lindahl, T Pan, Y-G Yang, C He
We report here that fat mass and obesity-associated protein (FTO) has efficient oxidative demethylation activity targeting the abundant N6-methyladenosine (m6A) residues in RNA in vitro. FTO knockdown with siRNA led to increased amounts of m6A in mRNA, whereas overexpression of FTO resulted in decreased amounts of m6A in human cells. We further show the partial colocalization of FTO with nuclear speckles, which supports the notion that m6A in nuclear RNA is a major physiological substrate of FTO.
RNA interference (RNAi) is the best way to effectively knock down gene expression to study protein function in a wide range of cell types.
2013
Authors: S Niland, B Ditkowski, D Parrandier, L Roth, H Augustin, JA Eble
Objective—The snake venom component rhodocetin-αβ (RCαβ) stimulates endothelial cell motility in an α2β1 integrin–independent manner. We aimed to elucidate its cellular and molecular mechanisms.
2012
Cellular delivery of polynucleotides by cationic cyclodextrin polyrotaxanes
Authors: P Dandekar, R Jain, M Keil, B Loretz, Leon Muijs, M Schneider, D Auerbach, G Jung, C-M Lehr, G Wenz
Cationic polyrotaxanes, obtained by temperature activated threading of cationic cyclodextrin derivatives onto water‐soluble cationic polymers (ionenes), form metastable nanometric polyplexes with pDNA and combinations of siRNA with pDNA. Because of their low toxicity, the polyrotaxane polyplexes constitute a very interesting system for the transfection of polynucleotides into mammalian cells. The complexation of Cy3-labeled siRNA within the polyplexes was demonstrated by fluorescence correlation spectroscopy. The uptake of the polyplexes (red) was imaged by confocal fluorescence microscopy using the A549 cell line as a model (blue: nuclei, green: membranes). The results prove the potential of polyrotaxanes for further investigations involving knocking down genes of therapeutic interest.
2012
MicroRNA-328 May Influence Myopia Development by Mediating the PAX6 Gene
Authors: K-C Chen, E Hsi1, C-Y Hu, W-W Chou, C-L Liang, S-H H Juo
We showed previously that single nucleotide polymorphism (SNP) rs662702 in PAX6 may be located in a microRNA-328 binding site that causes susceptibility to high myopia. Our study was done to elucidate the role of PAX6 and its relationship with microRNA-328 in myopia.
2012
A novel cell-penetrating peptide TAT-A1 delivers siRNA into tumor cells selectively
Authors: B Fang, L Jiang, M Zhang, FZ Ren
siRNA is promising in anti-tumor therapy. The main challenge is lack of tumor-specific intracellular delivery. In this study, a 6 amino acids peptide (A1) with high affinity for vascular endothelial growth factor receptor-1 (VEGFR1) was conjugated with a cell penetrating peptide (CPP) TAT to form a tumor-selective CPP. To evaluate the tumor-targeted penetrate property of TAT-A1, the uptake of TAT-A1 was measured by flow cytometry. The selectivity in vitro was tested in co-cultured tumor cells and normal cells by laser confocal microscope. The internalization efficiency of TAT-A1 was significantly higher than that of TAT (p < 0.05). TAT-A1 penetrated into tumor cells selectively when added to co-cultured tumor cells and normal cells due to the recognition of VEGFR1 which is over-expressed on tumor cells. Furthermore, siRNA was successfully transferred by TAT-A1 into tumor cells in a similar way of Lipofectamine 2000, which was proved to be an efficient vector. The knockout effect of siRNA transferred by TAT-A1 was obtained at both mRNA and protein level. These results indicated that the tumor-targeted TAT-A1 can act as an excellent vehicle for specific delivery of anti-cancer agents.
2012
MicroRNA-181b and microRNA-9 mediate arsenic-induced angiogenesis via NRP1
Authors: Y Cui, Z Han, Y Hu, G Song, C Hao, H Xia, X Ma
Environmental exposure to inorganic arsenic compounds has been reported to have serious health effects on humans. Recent studies reported that arsenic targets endothelial cells lining blood vessels, and endothelial cell activation or dysfunction, may underlie the pathogenesis of arsenic-induced diseases and developmental toxicity. It has been reported that microRNAs (miRNAs) may act as an angiogenic switch by regulating related genes. The present study was designed to test the hypothesis that arsenite-regulated miRNAs play pivotal roles in arsenic-induced toxicity.
2012
Authors: H ten Freyhaus, ES Calay, A Yalcin, SN Vallerie, L Yang, ZZ Calay, F Saatcioglu, GS Hotamisligil
The six-transmembrane protein Stamp2 plays an important role in metabolically triggered inflammation and insulin action. We report that Stamp2 is expressed in human and mouse macrophages, is regulated upon differentiation or activation, acts as an anti-inflammatory protein, and regulates foam cell formation. Absence of Stamp2 results in significant increases in cellular NADPH levels, and both NADPH homeostasis and the exaggerated inflammatory response of Stamp2−/− macrophages are rescued by exogenous wild-type but not by a reductase-deficient Stamp2 molecule.
2012
Authors: G Swaminathan, F Rossi, L-J Sierra, A Gupta, S Navas-Martín, J Martín-García
HIV-1 infection of macrophages plays a key role in viral pathogenesis and progression to AIDS. Polyinosine-polycytidylic acid (poly(I:C); a synthetic analog of dsRNA) and bacterial lipopolysaccharide (LPS), the ligands for Toll-like receptors (TLR) TLR3 and TLR4, respectively, are known to decrease HIV-1 infection in monocyte-derived macrophages (MDMs), but the mechanism(s) are incompletely understood.
2012
Authors: LM Litchfield, KA Riggs, AM Hockenberry, LD Oliver, KG Barnhart, J Cai, WM Pierce Jr, MM Ivanova, PJ Bates, SN Appana, S Datta, P Kulesza, J McBryan, CM Klinge
The orphan nuclear receptor COUP-TFII plays an < role in breast cancer. Previously we reported lower COUP-TFII expression in tamoxifen/endocrine- resistant versus sensitive breast cancer cell lines. The identification of COUP-TFII-interacting proteins will help to elucidate its mechanism of action as a transcriptional regulator in breast cancer.
2011
Authors: CY Hsieh, MJ Hsu, G Hsiao, YH Wang, CW Huang, SW Chen, T Jayakumar, PT Chiu, YH Chiu, JR Sheu
Recent studies have demonstrated that transcription factor nuclear factor (NF)-κB inhibition may contribute to the protective anti-inflammatory actions of andrographolide, an abundant component of plants of the genus Andrographis. However, the precise mechanism by which andrographolide inhibits NF-κB signaling remains unclear. We thus investigated the mechanism involved in andrographolide suppression of NF-κB signaling in rat vascular smooth muscle cells (VSMCs) exposed to proinflammatory stimuli, LPS, and IFN-γ. Andrographolide was shown to suppress LPS/IFN-γ-induced inducible nitric-oxide synthase and matrix metalloprotease 9 expression in rat VSMCs.
2011
Authors: J Antoine-Bertrand, A Ghogha, V Luangrath, FK Bedford, N Lamarche-Vane
The receptor Deleted in Colorectal Cancer (DCC) mediates the attractive response of axons to the guidance cue netrin-1 during development. On netrin-1 stimulation, DCC is phosphorylated and induces the assembly of signaling complexes within the growth cone, leading to activation of cytoskeleton regulators, namely the GTPases Rac1 and Cdc42.
2011
Authors: P Tarapore, Y Shu, P Guo, S-M Ho
Ovarian cancer is a highly metastatic and lethal disease, making it imperative to find treatments that target late-stage malignant tumors. The packaging RNA (pRNA) of bacteriophage phi29 DNA-packaging motor has been reported to function as a highly versatile vehicle to carry small interference RNA (siRNA) for silencing of survivin. In this article, we explore the potential of pRNA as a vehicle to carry siRNA specifically targeted to metallothionein-IIa (MT-IIA) messenger RNA (mRNA), and compare it to survivin targeting pRNA.
2011
Col V siRNA Engineered Tenocytes for Tendon Tissue Engineering
Authors: P Lu, GR Zhang, XH Song, XH Zou, LL Wang, HW Ouyang
The presence of uniformly small collagen fibrils in tendon repair is believed to play a major role in suboptimal tendon healing. Collagen V is significantly elevated in healing tendons and plays an important role in fibrillogenesis. The objective of this study was to investigate the effect of a particular chain of collagen V on the fibrillogenesis of Sprague-Dawley rat tenocytes, as well as the efficacy of Col V siRNA engineered tenocytes for tendon tissue engineering.
2011
Wnt Signaling Mediates Pathological Vascular Growth in Proliferative Retinopathy
Authors: RB Chalamalasetty, W C Dunty Jr, KK Biris, R Ajima, M Iacovino, A Beisaw, L Feigenbaum, DL Chapman, JK Yoon, M Kyba, TP Yamaguchi
Ischemic proliferative retinopathy, characterized by pathological retinal neovascularization, is a major cause of blindness in working-age adults and children. Defining the molecular pathways distinguishing pathological neovascularization from normal vessels is critical to controlling these blinding diseases with targeted therapy. Because mutations in Wnt signaling cause defective retinal vasculature in humans with some characteristics of the pathological vessels in retinopathy, we investigated the potential role of Wnt signaling in pathological retinal vascular growth in proliferative retinopathy.
2010
Authors: Z Yang, W Xiaohua, J Lei, T Ruoyun, X Mingxia, H Weichun, F Li, W Ping, Y Junwei
Urate is produced as the major end product of purine metabolism. In the last decade, the incidence of hyperuricemia increased markedly, and similar trends in the epidemiology of metabolic syndrome have been observed. Hyperuricemia is associated with renal disease, and recent studies have reported that mild hyperuricemia results in hypertension, intrarenal vascular disease, and renal injury. This has led to the hypothesis that uric acid may contribute to renal fibrosis and progressive renal disease.
2010
Evaluation of the effects of small interfering RNAs on in vitro replication of feline herpesvirus-1
Authors: RP Wilkes, SA Kania
To evaluate the ability of 5 small interfering RNAs (siRNAs) targeting mRNA of the feline herpesvirus-1 (FHV-1) DNA polymerase gene to reduce in vitro viral replication and gene expression of FHV-1, to evaluate combinations of these siRNAs with siRNAs that target the glycoprotein D gene of FHV-1, and to determine the combination or combinations of siRNAs that yield the greatest inhibition of in vitro viral replication.
2007
P-selectin primes leukocyte integrin activation during inflammation
Authors: H-B Wang, J-T Wang, L Zhang, ZH Geng, W-L Xu, T Xu, Y Huo, X Zhu, EF Plow, M Chen, J-G Geng
Selectins mediate leukocyte rolling and prime leukocytes for integrin-mediated leukocyte adhesion. However, neither the in vivo importance of nor the signaling pathway by which selectin-mediated integrin activation occurs has been determined. We report here that P-selectin-deficient mice manifested impaired leukocyte adhesion, which was 'rescued' by soluble P-selectin. Mechanistically, the cytoplasmic domain of P-selectin glycoprotein ligand 1 formed a constitutive complex with Nef-associated factor 1. After binding of P-selectin, Src kinases phosphorylated Nef-associated factor 1, which recruited the phosphoinositide-3-OH kinase p85-p110 heterodimer and resulted in activation of leukocyte integrins. Inhibition of this signal-transduction pathway diminished the adhesion of leukocytes to capillary venules and suppressed peritoneal infiltration of leukocytes. Our data demonstrate the functional importance of this newly identified signaling pathway mediated by P-selectin glycoprotein ligand 1.
2007
Authors: M Echevarrìa, AM Munõz-Cabello, R Sánchez-Silva, JJ Toledo-Aral, J López-Barneo
O2 is essential for aerobic life, and the classic view is that it diffuses freely across the plasma membrane. However, measurements of O2 permeability of lipid bilayers have indicated that it is much lower than previously thought, and therefore, the existence of membrane O2 channels has been suggested. We hypothesized that, besides its role as a water channel, aquaporin-1 (AQP-1) could also work as an O2 transporter, because this transmembrane protein appears to be CO2-permeable and is highly expressed in cells with rapid O2 turnover (erythrocytes and microvessel endothelium).
2006
Authors: JM Holaska, S Rais-Bahrami, KL Wilson
X-linked Emery-Dreifuss muscular dystrophy (X-EDMD) is inherited through mutations in emerin, a nuclear membrane protein. Emerin has proposed roles in nuclear architecture and gene regulation, but direct molecular links to disease were unknown. We report that Lim-domain only 7 (Lmo7) binds emerin directly with 125 nM affinity; the C-terminal half of human Lmo7 (hLmo7C) was sufficient to bind emerin in vitro. Lmo7 appeared relevant to EDMD because a deletion that removes Lmo7 (plus eight exons of a neighboring gene) in mice causes dystrophic muscles (1). Lmo7 localizes in the nucleus, cytoplasm and cell surface, particularly adhesion junctions (2). Our data suggest endogenous Lmo7 is a nucleocytoplasmic shuttling protein, and might also localize at focal adhesions in HeLa cells.
Ambion® mMESSAGE mMACHINE® High Yield Capped RNA Transcription Kits use Ambion's patented high-yield transcription technology for the routine synthesis of 15–35 μg of 7-methyl guanosine capped RNA from 1 μg of template DNA. Each kit includes sufficient reagents for 25 reactions.
2007
Prometaphase APC[cdh1] activity prevents non-disjunction in mammalian oocytes
Authors: A Reis, S Madgwick, H-Y Chang, I Nabti, M Levasseur, KT Jones
The first female meiotic division (meiosis I, MI) is uniquely prone to chromosome segregation errors through non-disjunction, resulting in trisomies and early pregnancy loss1. Here, we show a fundamental difference in the control of mammalian meiosis that may underlie such susceptibility.
Ambion® mMESSAGE mMACHINE® High Yield Capped RNA Transcription Kits use Ambion's patented high-yield transcription technology for the routine synthesis of 15–35 μg of 7-methyl guanosine capped RNA from 1 μg of template DNA. Each kit includes sufficient reagents for 25 reactions.
2012
Authors: WA Munoz, M Kloc, K Cho, M Lee, I Hofmann, A Sater, K Vleminckx, PD McCrea
The p120-catenin family has undergone a significant expansion during the evolution of vertebrates, resulting in varied functions that have yet to be discerned or fully characterized. Likewise, members of the plakophilins, a related catenin subfamily, are found throughout the cell with little known about their functions outside the desmosomal plaque.
Ambion® mMESSAGE mMACHINE® High Yield Capped RNA Transcription Kits use Ambion's patented high-yield transcription technology for the routine synthesis of 15–35 μg of 7-methyl guanosine capped RNA from 1 μg of template DNA.
2012
Pharmacological kinetics of BmK AS, a sodium channel site 4-specific modulator on Nav1.3
Authors: Zhi-Rui Liu, Jie Tao, Bang-Qian Dong, Gang Ding, Zhi-Jun Cheng, Hui-Qiong He, Yong-Hua Ji
In this study, the pharmacological kinetics of Buthus martensi Karsch (BmK) AS, a specific modulator of voltage-gated sodium channel site 4, was investigated on Nav1.3 expressed in Xenopus oocytes.
2012
Modulation of Gain-of-function α6*-Nicotinic Acetylcholine Receptor by β3 Subunits
Authors: B Dash, RJ Lukas
We previously have shown that β3 subunits either eliminate (e.g. for all-human (h) or all-mouse (m) α6β4β3-nAChR) or potentiate (e.g. for hybrid mα6hβ4hβ3- or mα6mβ4hβ3-nAChR containing subunits from different species) function of α6*-nAChR expressed in Xenopus oocytes, and that nAChR hα6 subunit residues Asn-143 and Met-145 in N-terminal domain loop E are important for dominant-negative effects of nAChR hβ3 subunits on hα6*-nAChR function. Here, we tested the hypothesis that these effects of β3 subunits would be preserved even if nAChR α6 subunits harbored gain-of-function, leucine- or valine-to-serine mutations at 9′ or 13′ positions (L9′S or V13′S) in their second transmembrane domains, yielding receptors with heightened functional activity and more amenable to assessment of effects of β3 subunit incorporation.
2011
Authors: P Silveyra, M Raval, B Simmons, S DiAngelo, G Wang, J Floros
Two human genes, SFTPA1 (SP-A1) and SFTPA2 (SP-A2), encode surfactant protein A, a molecule of innate immunity and surfactant-related functions. Several genetic variants have been identified for both genes. These include nucleotide (nt) polymorphisms, as well as alternative splicing patterns at the 5′ untranslated region (5′UTR).
pMIR-REPORT™ miRNA Expression Reporter Vector System
The Ambion® pMIR-REPORT™ miRNA Expression Reporter Vector System is for the measurement of miRNA expression in cells. It contains two mammalian expression vectors-—one for cloning miRNA targets and evaluating miRNA regulation, and the other for the normalization of transfection efficiency.
2012
Authors: N Patel, SM Tahara, P Malik, VK Kalra
Plasminogen activator inhibitor-1 (PAI-1) is a key physiological inhibitor of fibrinolysis. Previously, we reported placenta growth factor (PlGF) mediated transcriptional upregulation of PAI-1 mRNAexpression via activation of hypoxia-inducible factor-1α and activator protein-1 in human pulmonary microvascular endothelial cells (HPMVEC); which resulted in elevated PAI-1 in humans with sickle cell anemia (SCA).
mMESSAGE mMACHINE® T7 ULTRA Kit
The Ambion® mMESSAGE mMACHINE™ T7 Ultra Kit combines a new cap analog—Anti-Reverse Cap Analog (ARCA)—with Ambion's patented high-yield transcription technology, to generate RNA transcripts that produce higher protein yields compared to other transcripts upon translation. The kit includes sufficient reagents for 50 reactions.
2012
Replacement of H1 linker histone during bovine somatic cell nuclear transfer
Authors: Y Yun, GM Zhao, SJ Wu, W Li, AM Lei
Linker histone variants are involved in regulation of chromosome organization and gene transcription; several subtypes are expressed in the maturing oocyte and developing embryo. In Xenopus and mice, the transition between linker histone variants occurred following nuclear transfer, and apparently contributed to donor nuclear reprogramming. To determine whether such linker histone replacement occurred after bovine nuclear transfer, red fluorescent protein (RFP) tagged H1e (somatic linker histone H1e) donor cells and Venus tagged H1foo eggs were created, enucleated eggs were injected with donor cells, and embryos were created by fusion.
2008
Authors: Irina Y Tcherepanova, Melissa D Adams, Xiaorong Feng, Atsushi Hinohara, Joe Horvatinovich, David Calderhead, Don Healey and Charles A Nicolette
RNA transfection into dendritic cells (DCs) is widely used to achieve antigen expression as well as to modify DC properties. CD40L is expressed by activated T cells and interacts with CD40 receptors expressed on the surface of the DCs leading to Th1 polarization. Previous studies demonstrated that ectopic CD40L expression via DNA transfection into DCs can activate the CD40 receptor signal transduction cascade. In contrast to previous reports, this study demonstrates that the same effect can be achieved when RNA encoding CD40L is electroporated into DCs as evidenced by secretion of IL-12.
Life Technologies offers a wide variety of antibodies against key signaling proteins, cellular markers and organelles as well as cell junctions and CD markers.
2012
Tubule-specific ablation of endogenous β-catenin aggravates acute kidney injury in mice
Authors: D Zhou, Y Li, L Lin, L Zhou, P Igarashi, Y Liu
β-Catenin is a unique intracellular protein functioning as an integral component of the cell–cell adherens complex and a principal signaling protein mediating canonical Wnt signaling. Little is known about its function in adult kidneys in the normal physiologic state or after acute kidney injury (AKI). To study this, we generated conditional knockout mice in which the β-catenin gene was specifically disrupted in renal tubules (Ksp-β-cat−/−). These mice were phenotypically normal with no appreciable defects in kidney morphology and function. In the absence of β-catenin, γ-catenin functionally substituted for it in E-cadherin binding, thereby sustaining the integrity of epithelial adherens junctions in the kidneys. In AKI induced by ischemia reperfusion or folic acid, the loss of tubular β-catenin substantially aggravated renal lesions.
The last step in every RNA isolation protocol is to resuspend the purified RNA pellet. After painstakingly isolating the RNA, it is crucial that the pellet be suspended and stored in a safe, RNase-free solution.
2011
Label-free high-throughput microRNA expression profiling from total RNA
Authors: D Duan, K-x Zheng, Y Shen, R Cao, L Jiang, Z Lu, X Yan, J Li
MicroRNAs (miRNAs) are key biological regulators and promising disease markers whose detection technologies hold great potentials in advancing fundamental research and medical diagnostics. Currently, miRNAs in biological samples have to be labeled before being applied to most high-throughput assays.
The Ambion® Decade™ Marker System produces a set of radiolabeled RNA molecules of 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, and 10 nucleotides. The molecular weight markers are ideally suited for experiments where RNA molecules less than 150 nucleotides in length are being studied. Sufficient reagents are provided for 10 marker preparation reactions. This marker set is particularly useful for research laboratories studying tRNA, snRNA, snoRNA, miRNA, siRNA and other small RNA molecules.
2012
Use of Northern Blotting for Specific Detection of Small RNA Molecules in Transgenic Plants
Authors: B Khraiwesh
Small RNAs (20–24 nucleotides long and nonprotein coding) have been increasingly investigated. They are responsible for phenomena described as RNA interference (RNAi), cosuppression, gene silencing, or quelling. Major classes of small RNAs include microRNAs (miRNAs) and small interfering RNAs (siRNAs), which differ in their biosynthesis. MiRNAs control the expression of cognate target genes by binding to reverse complementary sequences, resulting in cleavage or translational inhibition of the target RNA. SiRNAs have similar structure, function, and biogenesis as miRNAs; siRNAs derive from long double-stranded RNA of transgenes, endogenous repeat sequences, or transposons. Understanding these fundamental processes requires the sensitive and specific detection of small RNA species. In this report, we present a simple Northern blot protocol for small RNAs in transgenic plants.
Amino Allyl MessageAmp™ II aRNA Amplification Kit
The Ambion® Amino Allyl II MessageAmp™ aRNA Amplification Kit includes reagents for aRNA amplification with amimo allyl NTP incorporation. The kit includes sufficient reagents for 20 reactions. Cy™ dyes are not included.
2011
Authors: B Pouvreau, S Baud, V Vernoud, V Morin, C Py, G Gendrot, J-P Pichon, J Rouster, W Paul,PM Rogowsky
WRINKLED1 (WRI1), a key regulator of seed oil biosynthesis in Arabidopsis (Arabidopsis thaliana), was duplicated during the genome amplification of the cereal ancestor genome 90 million years ago. Both maize (Zea mays) coorthologs ZmWri1a and ZmWri1b show a strong transcriptional induction during the early filling stage of the embryo and complement the reduced fatty acid content of Arabidopsis wri1-4 seeds, suggesting conservation of molecular function.
Illumina® TotalPrep™ RNA Amplification Kit
Ambion®'s Illumina® TotalPrep™ RNA Amplification Kit (patent pending) is a complete system for generating biotinylated, amplified RNA for hybridization with Illumina Sentrix® arrays. The kit have significantly increased sensitivity, providing increased Percent Present calls and single round amplification with just 50 ng of input RNA.
2011
Anthrax Lethal Toxin-Induced Gene Expression Changes in Mouse Lung
Authors: EK Dumas, PM Cox, C O’Connor Fullenwider, M Nguyen, M Centola, MB Frank, I Dozmorov, JA James, AD Farris
A major virulence factor of Bacillus anthracis is the anthrax Lethal Toxin (LeTx), a bipartite toxin composed of Protective Antigen and Lethal Factor. Systemic administration of LeTx to laboratory animals leads to death associated with vascular leakage and pulmonary edema. In this study, we investigated whether systemic exposure of mice to LeTx would induce gene expression changes associated with vascular/capillary leakage in lung tissue. We observed enhanced susceptibility of A/J mice to death by systemic LeTx administration compared to the C57BL/6 strain. LeTx-induced groups of both up- and down-regulated genes were observed in mouse lungs 6 h after systemic administration of wild type toxin compared to lungs of mice exposed to an inactive mutant form of the toxin. Lungs of the less susceptible C57BL/6 strain showed 80% fewer differentially expressed genes compared to lungs of the more sensitive A/J strain.
This kit is a major improvement over the basic rapid amplification of cDNA ends (RACE) protocol. The RLM-RACE procedure selects only full-length mRNA — no rRNA, tRNA or degraded RNA — and facilitates the cloning of sequences from the 5' ends of messages.
2013
Authors: S Rana, SK Lattoo, N Dhar, S Razdan, WW Bhat, RS Dhar, R Vishwakarma
Withania somnifera (L.) Dunal, a highly reputed medicinal plant, synthesizes a large array of steroidal lactone triterpenoids called withanolides. Although its chemical profile and pharmacological activities have been studied extensively during the last two decades, limited attempts have been made to decipher the biosynthetic route and identification of key regulatory genes involved in withanolide biosynthesis.
2012
Authors: L Zhai, Z Liu, X Zou, Y Jiang, F Qiu, Y Zheng, Z Zhang
MicroRNAs (miRNAs) are critical post-transcriptional modulators of gene expression involving in plant responses to abiotic stress. However, the regulation of miRNA in the morphological response to waterlogging is poorly understood in maize. In this study, we detected miRNAs and their targets that expressed in waterlogged crown roots of maize seedlings in two inbred lines (Hz32 and Mo17) by RNA sequencing.
2011
Anthrax Lethal Toxin-Induced Gene Expression Changes in Mouse Lung
Authors: EK Dumas, PM Cox, C O’Connor Fullenwider, M Nguyen, M Centola, MB Frank, I Dozmorov, JA James, AD Farris
A major virulence factor of Bacillus anthracis is the anthrax Lethal Toxin (LeTx), a bipartite toxin composed of Protective Antigen and Lethal Factor. Systemic administration of LeTx to laboratory animals leads to death associated with vascular leakage and pulmonary edema. In this study, we investigated whether systemic exposure of mice to LeTx would induce gene expression changes associated with vascular/capillary leakage in lung tissue. We observed enhanced susceptibility of A/J mice to death by systemic LeTx administration compared to the C57BL/6 strain. LeTx-induced groups of both up- and down-regulated genes were observed in mouse lungs 6 h after systemic administration of wild type toxin compared to lungs of mice exposed to an inactive mutant form of the toxin. Lungs of the less susceptible C57BL/6 strain showed 80% fewer differentially expressed genes compared to lungs of the more sensitive A/J strain.
2009
Authors: M Knight, N Raghavan, C Goodall, C Cousin, W Ittiprasert, A Sayed, A Miller, DL Williams, CJ Bayne
To identify gene(s) that may be associated with resistance/susceptibility in the intermediate snail host Biomphalaria glabrata to Schistosoma mansoni infection, a snail albumen gland cDNA library was differentially screened and a partial cDNA encoding an antioxidant enzyme thioredoxin peroxidase (Tpx), or peroxiredoxin (Prx), was identified. The 753 bp full-length, single-copy, constitutively expressed gene now referred to as BgPrx4 was later isolated.
MagMAX™-96 Viral RNA Isolation Kit
Ambion's MagMAX™ Viral RNA isolation kits have been optimized for use with biological fluids and cell-free samples such as serum, plasma, swabs, and cell culture media.
2011
Authors: F Sun, PJ Ferro, B Lupiani, J Kahl, ME Morrow, JP Flanagan, C Estevez, A Clavijo
The Reticuloendotheliosis virus (REV) group of retroviruses infects a wide range of avian species, including chickens, turkeys, ducks, geese, quail, and prairie chickens. The objective of the present study was to develop a highly sensitive and specific diagnostic test for the detection of REV in whole blood samples. In order to increase the diagnostic sensitivity, a duplex real-time polymerase chain reaction (PCR) that detects both the envelope protein gene (env) and the long terminal repeat (LTR) region of REV was designed. This assay demonstrated greater analytical and diagnostic sensitivity than the gel-based PCR assay when using DNA extracted from whole blood by both phenol-chloroform and magnetic bead methods. In general, threshold cycle values in the duplex real-time PCR assay were lower from DNA extracted using the magnetic bead system compared to DNA extracted by the phenol-chloroform method. Data presented herein show the successful development of a rapid and accurate test procedure, with high-throughput capability, for the diagnosis of REV infection using avian blood samples.
The mirVana™ PARIS Kit is for the purification of both native protein and RNA (including small RNA) from the same sample. Each kit contains sufficient reagents and consumables for either 40 isolations of protein and total RNA (including small RNAs), or 20 isolation of protein and separate large and small RNA fractions.
2012
Authors: H Song, Q Wang, J Wen, S Liu, X Gao, J Cheng, D Zhang
Fibrodysplasia ossificans progressiva (FOP) is a rare congenital disorder of skeletal malformations and progressive extraskeletal ossification. There is still no effective treatment for FOP. All FOP individuals harbor conserved point mutations in ACVR1 gene that are thought to cause ACVR1 constitutive activation and activate BMP signal pathway. The constitutively active ACVR1 is also found to be able to cause endothelial-to-mesenchymal transition (EndMT) in endothelial cells, which may cause the formation of FOP lesions. MicroRNAs (miRNAs) play an essential role in regulating cell differentiation. Here, we verified that miR-148a directly targeted the 3' UTR of ACVR1 mRNA by reporter gene assays and mutational analysis at the miRNA binding sites, and inhibited ACVR1 both at the protein level and mRNA level. Further, we verified that miR-148a could inhibit the mRNA expression of the Inhibitor of DNA binding (Id) gene family thereby suppressing the BMP signaling pathway. This study suggests miR-148a is an important mediator of ACVR1, thus offering a new potential target for the development
of therapeutic agents against FOP.
2012
Authors: C Vidal, S Bermeo, W Li, D Huang, R Kremer, G Duque*
Interferon gamma (IFNγ) has been reported to induce osteoblastogenesis from mesenchymal stem cells (MSCs) both in vitro and in vivo. With ageing, adipocytes outnumber osteoblasts within the bone microenvironment leading to a decrease in bone formation. Since both osteoblasts and adipocytes are of mesenchymal origin, we hypothesized that IFNγ treatment might negatively affect adipogenesis while stimulating osteoblastogenesis in human MSC.
The mirVana™ miRNA Isolation Kit uses a rapid procedure to isolate small RNAs from tissue and cells using an efficient glass fiber filter (GFF) – based method. The method isolates total RNA ranging in size from kilobases down to 10-mers.
2012
Authors: DW Lee, M Futami, M Carroll, Y Feng, Z Wang, M Fernandez, Z Whichard, Y Chen, S Kornblau, EJ Shpall, CE Bueso-Ramos, SJ Corey
The myelodysplastic syndromes (MDSs) comprise a group of disorders characterized by multistage progression from cytopenias to acute myeloid leukemia (AML). They display exaggerated apoptosis in early stages, but lose this behavior during evolution to AML. The molecular basis for loss of apoptosis is unknown. To investigate this critical event, we analyzed phosphatidylinositol (PI) 3′kinase signaling, implicated as a critical pathway of cell survival control in epithelial and hematological malignancies.
2012
Authors: J Bao, D Li, L Wang, J Wu, Y Hu, Z Wang, Y Chen, X Cao, C Jiang, W Yan, C Xu
MicroRNAs (miRNAs) mainly function as post-transcriptional regulators and are involved in a wide range of physiological and pathophysiological processes such as cell proliferation, differentiation, apoptosis, and tumorigenesis. Mouse testes express a large number of miRNAs. However, the physiological roles of these testicular miRNAs remain largely unknown.
2012
An atlas of DNA methylomes in porcine adipose and muscle tissues
Authors: M Li, H Wu, Z Luo, Y Xia et al
It is evident that epigenetic factors, especially DNA methylation, have essential roles in obesity development. Here, using pig as a model, we investigate the systematic association between DNA methylation and obesity. We sample eight variant adipose and two distinct skeletal muscle tissues from three pig breeds living within comparable environments but displaying distinct fat level.
2012
MicroRNA-409-3p regulates cell proliferation and apoptosis by targeting PHF10 in gastric cancer
Authors: C Li, H Nie, M Wang, L Su, J Li, B Yu, M Wei, J Ju, Y Yu, M Yan, Q Gu, Z Zhu, B Liu
Emerging evidence has indicated microRNAs are involved in tumor development and progression, acting as tumor suppressors or oncogenes. Here we report that miR-409-3p was significantly downregulated in gastric cancer (GC) cell lines and tissues. Overexpression of miR-409-3p in SGC-7901 gastric cancer cells dramatically suppressed cell proliferation and induced cell apoptosis both in vitro and in vivo. Furthermore, we demonstrate that the transcriptional regulator PHF10 was a target of miR-409-3p. Taken together, these findings suggest that miR-409-3p may function as a novel tumor suppressor in GC and its anti-oncogenic activity may involve the direct targeting and inhibition of PHF10.
2012
Colorectal Cancer Migration and Invasion Initiated by microRNA-106α
Authors: B Feng, TT Dong, L L Wang, HM Zhou, HC Zhao, F Dong, MH Zheng
MicroRNAs have been implicated in the regulation of several cellular signaling pathways of colorectal cancer (CRC) cells. Although emerging evidence proves that microRNA (miR)-106a is expressed highly in primary tumor and stool samples of CRC patients; whether or not miR-106a mediates cancer metastasis is unknown. We show here that miR-106a is highly expressed in metastatic CRC cells, and regulates cancer cell migration and invasion positively in vitro and in vivo.
2012
MiR-138 induces cell cycle arrest by targeting cyclin D3 in hepatocellular carcinoma
Authors: W Wang, L-J Zhao, Y-X Tan, H Ren, Z-T Qi
It has been reported that decreased Dicer expression leads to Alu RNAs accumulation in human retinal pigmented epithelium cells, and Dicer may process the endogenous SINE/B1 RNAs (the rodent equivalent of the primate Alu RNAs) into small interfering RNAs (siRNAs). In this study, we aimed to address whether Dicer can process Alu RNAs and their common ancestor, 7SL RNA. Using Solexa sequencing technology, we showed that Alu-derived small RNAs accounted for 0.6% of the total cellular small RNAs in HepG2.2.15 cells, and the abundance decreased when Dicer was knocked down.
2012
Pri-miR-17-92a transcript folds into a tertiary structure and autoregulates its processing
Authors: S Chakraborty, S Mehtab, A Patwardhan, Y Krishnan
MicroRNAs control gene expression either by RNA transcript degradation or translational repression. Expressions of miRNAs are highly regulated in tissues, disruption of which leads to disease. How this regulation is achieved and maintained is still largely unknown.
2012
Novel Pathways in the Pathobiology of Human Abdominal Aortic Aneurysms
Authors: I Hinterseher, R Erdman, JR Elmore, E Stahl, MC Pahl, K Derr, A Golden, JH Lillvis, MC Cindric, K Jackson, WD Bowen, CM Schworer, MA Chernousov, DP Franklin, JL Gray, RP Garvin, Z Gatalica, DJ Carey, G Tromp, H Kuivaniemi
Abdominal aortic aneurysm (AAA), a dilatation of the infrarenal aorta, typically affects males 1 65 years. The pathobiological mechanisms of human AAA are poorly understood. The goal of this study was to identify novel pathways involved in the development of AAAs. A custom-designed ‘AAA-chip’ was used to assay 43 of the differentially expressed genes identified in a previously published microarray study between AAA (n = 15) and control (n = 15) infrarenal abdominal aorta. Protein analyses were performed on selected genes.
2012
Csy4 relies on an unusual catalytic dyad to position and cleave CRISPR RNA
Authors: RE Haurwitz, SH Sternberg, JA Doudna
CRISPR-Cas adaptive immune systems protect prokaryotes against foreign genetic elements. crRNAs derived from CRISPR loci base pair with complementary nucleic acids, leading to their destruction. In Pseudomonas aeruginosa, crRNA biogenesis requires the endoribonuclease Csy4, which binds and cleaves the repetitive sequence of the CRISPR transcript. Biochemical assays and three co-crystal structures of wild-type and mutant Csy4/RNA complexes reveal a substrate positioning and cleavage mechanism in which a histidine deprotonates the ribosyl 2'-hydroxyl pinned in place by a serine, leading to nucleophilic attack on the scissile phosphate.
2011
Repertoire of Porcine MicroRNAs in Adult Ovary and Testis by Deep Sequencing
Authors: M Li, Y Liu, T Wang et al
MicroRNAs (miRNAs), a large family of short endogenous RNAs known to post-transcriptionally repress gene expression, participate in the regulation of almost every cellular process. Changes in miRNA expression are associated with many pathologies. Ovarian folliculogenesis and testicular spermatogenesis are complex and coordinated biological processes, in which tightly regulated expression and interaction of a multitude of genes could be regulated by these miRNAs. Identification and preliminary characterization of gonad-specific miRNAs would be a prerequisite for a thorough understanding of the role that miRNA-mediated posttranscriptional gene regulation plays in mammalian reproduction.
2011
Circulating microRNAs in hepatitis B virus–infected patients
Authors: F Ji, B Yang, X Peng, H Ding, H You, P Tien
MicroRNAs (miRNAs) are stably present in human serum. The relationship between circulating miRNAs and hepatitis B virus (HBV) infected liver disease has not been previously reported. Applied Biosystems array-based miRNA expression profiling was performed on pooled sera obtained from identified groups of chronic asymptomatic carriers (ASC), patients with chronic hepatitis B (CHB) and HBV-associated acute-on-chronic liver failure (ACLF), as well as healthy controls (HC).
2011
Authors: JA López, LM Alvarez-Salas
MicroRNAs (miRNA) regulate expression of several genes associated with human cancer. Here, we analyzed the function of miR-34c, an effector of p53, in cervical carcinoma cells. Expression of either miR-34c-3p or miR-34c-5p mimics caused inhibition of cell proliferation in the HPV-containing SiHa cells but not in other cervical cells irrespective of tumorigenicity and HPV content.
2010
Authors: H Wei, C Wang, C Zhang, P Li, F Wang, Z Zhang
Neural stem cells’ transplantation has been proposed as a future therapy for spinal cord injury. The challenge is how to make proportionally more neural stem cells differentiate into spinal motor neurons. Recent reports reveal that microRNAs play an important role in regulating stem cell self-renewal and differentiation.
2010
Authors: N Bhatnagar, X Li, SKR Padi, Q Zhang, M-s Tang, B Guo
Advanced prostate cancers are known to acquire not only invasive capabilities but also significant resistance to chemotherapy-induced apoptosis. To understand how microRNAs (miRNAs) may contribute to prostate cancer resistance to apoptosis, we compared microRNA expression profiles of a benign prostate cancer cell line WPE1-NA22 and a highly malignant WPE1-NB26 cell line (derived from a common lineage). We found that miR-205 and miR-31 are significantly downregulated in WPE1-NB26 cells, as well as in other cell lines representing advanced-stage prostate cancers. Antiapoptotic genes BCL2L2 (encoding Bcl-w) and E2F6 are identified as the targets of miR-205 and miR-31, respectively. By downregulating Bcl-w and E2F6, miR-205 and miR-31 promote chemotherapeutic agents-induced apoptosis in prostate cancer cells.
