Silencer® Select siRNAs for Non-coding RNAs
High Performance, Naturally
What is ncRNA?
Non-coding RNAs (ncRNAs) are RNAs that are transcribed from the genome but are not translated into proteins. Recent data suggest that thousands of long (>100 nt) ncRNAs are expressed in a developmentally regulated manner in mammals. Growing evidence also indicates that these ncRNA transcripts are functional, especially in the regulation of epigenetic processes, and thus ncRNAs are emerging as important regulators of diverse functions. ncRNAs represent a new frontier of molecular genetic, molecular biological, physiological, and cell biological research with tremendous potential to advance our understanding of the biological processes in human health and disease.
Silencer® Select siRNAs for ncRNA
Ambion® Silencer® Select siRNAs for non-coding RNA (ncRNA) now enable ncRNA researchers for the first time to easily obtain siRNAs for any ncRNA. The same industry-leading siRNA design and chemical modifications used in our standard Silencer® Select siRNAs are also incorporated into Silencer® Select siRNAs for ncRNA (Figure 1). Pre-designed siRNAs for ncRNA are designed to target both cytoplasmic and nuclear long (>100 nt) ncRNAs in the NCBI and RNAdb databases for human, mouse, and rat. The ncRNAs for which pre-designed siRNA are available closely match the TaqMan® Non-coding RNA Assays currently offered by Life Technologies. Additional siRNAs can be custom designed for these targets and other newer targets using the custom design feature. Up to 10 pre-designed siRNAs are displayed for each ncRNA target.
For information or inquiries for siRNA libraries targeting non-coding RNAs in human. mouse or rat, please contact RNAilibraries@lifetech.com
Figure 1. The concept behind Silencer® Select siRNAs.
Strand-Specific Knockdown of ncRNA Transcripts Using Silencer® Select siRNAs
Silencer® Select siRNAs are designed with off-target checks performed against the coding (NM and XM) and non-coding (NR and XR) transcripts in the NCBI RefSeq database to ensure that siRNA designs are specific for the intended target. LNA® chemical modifications were incorporated at carefully selected positions to further enhance specificity. The use of Silencer® Select siRNAs offers tremendous advantages, especially when targeting transcripts that may be transcribed in both sense and antisense orientation. Because a high level of antisense and sense transcription is estimated to occur, strand-specific knockdown is an important feature that adds significant value to any siRNA experiment.
For example, the ncRNA MCM3APAS is a natural antisense transcript of MCM3AP (Figure 2). Several Silencer® Select siRNAs were designed against MCM3APAS and tested for knockdown. The expression of MCM3AP was also assessed. All the Silencer® Select siRNAs targeted to MCM3APAS specifically knocked down only the MCM3APAS transcript and not the MCM3AP transcript (Figure 3). Moreover, although siRNA 7, which is designed to a region that is common to both the MCM3APAS and MCM3AP transcripts, could have been effective against both transcripts, only the guide strand exhibited activity and led to knockdown of MCM3APAS. The passenger strand is completely inactive due to the Silencer® Select chemical modifications, and no knockdown of MCM3AP was observed.
Figure 2. Alignment of MCM3AP and MCM3APAS transcripts. MCM3AP (NM-003906.3) and MCM3APAS (NR-002776.3) transcript alignment shows that their genome coordinates overlap and the transcripts are natural sense and antisense transcripts. The sequence alignment below shows siRNA 7 targets both the transcripts.
Figure 3. Specificity of LNA-modified Silencer® Select siRNAs. Multiple Silencer® Select siRNAs designed to knock down non-coding RNA MCM3APAS (anti-sense transcript) effectively knock down the intended ncRNA target, but not the coding MCM3AP (sense transcript) in HeLa cells.
Case Study: Robust Knockdown of MALAT-1, a Nuclear Localized ncRNA, Using Multiple siRNAs Designed Using the Ambion® Silencer® Select Pipeline
Metastasis-associated lung adenocarcinoma transcript 1 (MALAT-1) is a long ncRNA expressed from chromosome 11 and is known to be misregulated in several human carcinomas. MALAT-1 is a bona fide nuclear localized ncRNA, and its overexpression is implicated in the development and progression of numerous malignant cancers.
Using SOLiDTM System next-generation sequencing technology for deep sequencing, we have identified MALAT-1 as one of the highly expressed ncRNAs in HeLa cervical cancer cells. We have also confirmed the expression of MALAT-1 in several common human cell lines (HeLa, A549, HEK293, Jurkat, U2-OS, MCF7, and HuH7) at high levels using the MALAT-1 specific non-coding TaqMan® Assay (Hs00273907_s1) in quantitative real-time PCR (qRT-PCR).
We used the Ambion® Silencer® Select pipeline to design siRNAs and tested in HeLa and A549 cells to inhibit MALAT-1 expression and evaluated the knockdown efficacy using the LipofectamineTM RNAiMAX Transfection Reagent. Our data from HeLa cells showed that the siRNAs are effective at 30 nM to silence MALAT-1 by 80% or greater, and this effect occurred as early as 24 hours post-transfection and persisted up to 5 days. The knockdown was further confirmed by northern blot analysis, which showed a strong reduction in MALAT-1 transcript levels that is consistent with the knockdown determination by qRT-PCR (Figure 4).
Figure 4. Robust knockdown of nuclear localized MALAT-1 ncRNA in HeLa cells by multiple Silencer® Select siRNAs and confirmation of results by northern analysis.