Isolation to Detection: RNA Analysis with Low Copy Number Templates
| Together, Ambion and Applied Biosystems (AB) provide researchers with a complete family of products that takes experiments from sample preparation through target detection and analysis. For limited samples and small sample sizes, RNA can be extracted from numerous small tissue samples with one of several nucleic acid isolation kits from Ambion (see table). Quantitative analysis of gene expression can then be assessed using RT-PCR reagents and instruments from Applied Biosystems (Figure 1).  |
RNA Isolation
Ambion, The RNA Company®, has a complete line of RNA isolation products that addresses a wide range of samples and applications.
MELT™ Total Nucleic Acid Isolation System (patent pending) provides hands-free tissue digestion that takes only minutes—no grinding or use of a Polytron® homogenizer required—making MELT ideal for processing samples for downstream high throughput amplification.
MagMAX™-96 Total RNA Isolation System is optimized for high throughput isolation of RNA from 25 cells to 2 x 106 cells, as well as small plant and mammalian tissue samples (up to 10 mg). This magnetic bead-based technology binds RNA more efficiently than glass fiber filter methods, resulting in higher RNA yields.
RNAqueous®-Micro Kit is optimized for the purification of total RNA from micro-sized samples such as 10 to 5 x 105 cultured cells, as few as 10 microdissected cells, or up to 10 mg tissue. Samples are solubilized in a powerful chaotropic solution, and RNA is purified on a glass fiber filter cartridge specifically designed for very small samples. A simple modification of the protocol allows recovery of both large and small RNA species including tRNAs, 5S rRNA, and microRNAs.
RNAqueous®-4PCR Kit is designed to isolate RNA free of genomic DNA contamination from very small samples such as needle biopsies and small numbers of isolated cells (as little as 1 mg of tissue or 100 cells).
MELT™ Total Nucleic Acid Isolation System (patent pending) provides hands-free tissue digestion that takes only minutes—no grinding or use of a Polytron® homogenizer required—making MELT ideal for processing samples for downstream high throughput amplification.
MagMAX™-96 Total RNA Isolation System is optimized for high throughput isolation of RNA from 25 cells to 2 x 106 cells, as well as small plant and mammalian tissue samples (up to 10 mg). This magnetic bead-based technology binds RNA more efficiently than glass fiber filter methods, resulting in higher RNA yields.
RNAqueous®-Micro Kit is optimized for the purification of total RNA from micro-sized samples such as 10 to 5 x 105 cultured cells, as few as 10 microdissected cells, or up to 10 mg tissue. Samples are solubilized in a powerful chaotropic solution, and RNA is purified on a glass fiber filter cartridge specifically designed for very small samples. A simple modification of the protocol allows recovery of both large and small RNA species including tRNAs, 5S rRNA, and microRNAs.
RNAqueous®-4PCR Kit is designed to isolate RNA free of genomic DNA contamination from very small samples such as needle biopsies and small numbers of isolated cells (as little as 1 mg of tissue or 100 cells).
Reverse Transcription
The High-Capacity cDNA Reverse Transcription Kits for 200 and 1000 reactions contain reagents for reverse transcription (RT) of total RNA to single-stranded cDNA. While designed for short or long term archival of cDNA, the kit yields very quantitative amplification of total RNA from 0.02 to 2 mg of total RNA.The resultant cDNA is ideal for:
- Short-term and long-term archival storage
- Quantitative PCR
- Conversion to cRNA
NEW--Sample Preamplification
Before quantitative, real-time PCR, use the TaqMan® PreAmp Master Mix Kit to enrich cDNA for specific gene targets. This preamplification process is uniform and does not introduce bias, so this kit is ideal for rare or small samples (for example, laser capture microdissection, formalin-fixed paraffin-embedded tissues, needle biopsies, and single cells).
Target Amplification
Pre-designed, pre-optimized TaqMan® Gene Expression Assays are a comprehensive collection of over 700,000 probe and primer sets for quantitative gene expression analysis of human, mouse, rat, Arabidopsis, and Drosophila genes. These assays let you quickly, easily, and affordably perform precise gene expression studies using real-time PCR.
Detection
The Applied Biosystems 7300, 7500, 7500 Fast, and 7900HT Real-Time PCR systems offer you a portfolio of powerful and versatile platforms for real-time PCR detection. The 7500 Fast system’s high speed performance delivers results in 30 minutes.
(This figure is referenced in the sidebar text.)
(This figure is referenced in the sidebar text.)
Small Samples—Big Results—A Better Choice
Ambion’s MagMAX™ Total RNA Isolation Kit delivers high quality RNA to maximize the success of your gene expression studies. The kit is optimized for high throughput isolation of RNA from 25 to 2 x 106 cells, as well as small plant and mammalian tissue samples (up to 10 mg). Ambion’s TURBO DNase™ is included to enhance genomic DNA removal. The quantitative RT-PCR results (see figure at bottom of page) demonstrate the excellent linearity obtained with this kit when total RNA was isolated from increasing numbers of K562 cells. The quality and purity of the eluted RNA make it suitable for most common downstream application, such as qRT-PCR, microarray analysis, Northern blotting, and ribonuclease protection assays (RPA).
