Silencer® Select siRNAs

  • Incorporate the latest improvements in siRNA design, off-target effect prediction algorithms, and chemistry
  • Provide unrivalled silencing consistency, potency, and specificity
  • Result in fewer failed experiments due to poor silencing
  • Yield cleaner, more consistent phenotypic data

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 func- tional, 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.

Strand-specific knockdown of ncRNA transcripts using Silencer® Select siRNAs

Silencer® Select siRNAs are designed with off-target checks per- formed 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® chemi- cal modifications were incorporated at carefully selected posi- tions to further enhance specificity. The use of Silencer® Select siRNAs offers tremendous advantages, especially when tar- geting 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 knock- down 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 specif- ically 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.

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 technol- ogy 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 analy- sis, which showed a strong reduction in MALAT-1 transcript levels that is consistent with the knockdown determination by qRT-PCR (Figure 4).