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Silencer Select siRNAs for Long Non-Coding RNAs
Long non-coding RNAs (lncRNAs) are a type of non-coding RNAs (ncRNAs), typically over 200 nt, that are abundant in the mammalian transcriptome. LncRNAs have been shown to regulate cell functions including cell signaling, tumor progression, and metabolic regulation. Knockdown of lncRNAs with siRNA is a useful technique to determine the function and significance of a particular lncRNA. However, this can be challenging due to their cellular location and secondary structure. To help overcome this, Silencer Select lncRNA siRNAs for lncRNA include the following:
- Locked nucleic acid (LNA) technology helps ensure high specificity and helps reduce off-target effects
- Silencer Select technology designs highly potent siRNA with low cell toxicity for more accurate results
Features of Silencer Select siRNAs targeting lncRNAs
- Comprehensive portfolio of Silencer Select siRNAs targeting over 5000 lncRNA targets
- Validated using databases including Gencode v27 for optimal coverage of lncRNA loci, pseudogene loci, and alternatively spliced transcripts
- Pre-designed siRNAs closely match existing TaqMan non-coding assays
Tips for success
lncRNAs typically have lower expression levels compared to coding genes and expression can vary across cell lines. Improve results for knockdown of lncRNA with these suggestions:
- Verify expression levels in your cells of interest prior to your experiment.
- Choose at least three siRNAs for each lncRNA of interest.
- Use locked nucleic acid (LNA) modified siRNA (e.g. Silencer Select siRNA) over unmodified or earlier-generation siRNA for greater potency and lower off-target effects.
- Use a reliable transfection reagent such as Invitrogen Lipofectamine RNAiMAX Transfection Reagent for high transfection efficiency and improved cell viability.
Efficient knockdown of lncRNAs using Silencer Select siRNA
Results of targeted knockdown in HeLa and HEK293T cells indicate that two or more siRNA effected at least 50% knockdown. 80% knockdown or more was obtained for six out of the seven targets. Knockdown was effective for lncRNA with nuclear or cytoplasmic localization.
Figure 1. Five different siRNAs were tested per gene at a concentration of 25 nM. Transfections were done in duplicates and each siRNA was delivered into 1X104 HeLa or HEK293T cells in a 96-well format using Lipofectamine RNAiMAX Transfection Reagent. Cells were harvested 48 hours post-transfection followed by cDNA synthesis and gene expression profiling using TaqMan Gene Expression Cells-to-CT Kit and TaqMan Real-Time PCR assays. TaqMan qPCR assays were performed in triplicates and remaining expression was normalized to a scrambled (Scr siRNA) negative control (Scr) siRNA transfected sample.
Cells maintain high cell viability post siRNA transfection
Transfection reagents alone or in combination with siRNAs may lead to a nonspecific decrease in cell viability. This may impact interpretation of knockdown results. Five different siRNA against seven different lncRNA were transfected into the indicated cells. No significant decrease in viability was detected.
Figure 2. 25 nM of each siRNA was used to transfect 1X104 HeLa or HEK293T cells in 96-well plate format using Lipofectamine RNAiMAX transfection reagent. Forty-eight hours post transfection and prior to performing TaqMan real-time PCR assay cell viability was analyzed using the PrestoBlue Cell Viability assay. Percent cell viability was normalized to samples treated with Lipofectamine RNAiMAX transfection reagent without any siRNA.
Case study: Robust knockdown of MALAT-1, a nuclear localized lncRNA, with multiple siRNAs designed using the Ambion Silencer Select pipeline
Metastasis-associated lung adenocarcinoma transcript 1 (MALAT-1) is a lncRNA expressed from chromosome 11 and is known to be mis-regulated in several human carcinomas. MALAT-1 lncRNA is expressed in the nucleus, and its overexpression is implicated in the development and progression of numerous malignant cancers.
MALAT-1 expression was confirmed 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).
siRNAs designed against MALAT-1 were transfected into HeLa and A549 cells using Lipofectamine RNAiMAX Transfection Reagent. Knockdown was evaluated by real time PCR. 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 3).
Figure 3. Robust knockdown of nuclear localized MALAT-1 ncRNA in HeLa cells by multiple Silencer Select siRNAs and confirmation of results by northern analysis.
Tips for success
lncRNAs typically have lower expression levels compared to coding genes and expression can vary across cell lines. Improve results for knockdown of lncRNA with these suggestions:
- Verify expression levels in your cells of interest prior to your experiment.
- Choose at least three siRNAs for each lncRNA of interest.
- Use locked nucleic acid (LNA) modified siRNA (e.g. Silencer Select siRNA) over unmodified or earlier-generation siRNA for greater potency and lower off-target effects.
- Use a reliable transfection reagent such as Invitrogen Lipofectamine RNAiMAX Transfection Reagent for high transfection efficiency and improved cell viability.
Efficient knockdown of lncRNAs using Silencer Select siRNA
Results of targeted knockdown in HeLa and HEK293T cells indicate that two or more siRNA effected at least 50% knockdown. 80% knockdown or more was obtained for six out of the seven targets. Knockdown was effective for lncRNA with nuclear or cytoplasmic localization.
Figure 1. Five different siRNAs were tested per gene at a concentration of 25 nM. Transfections were done in duplicates and each siRNA was delivered into 1X104 HeLa or HEK293T cells in a 96-well format using Lipofectamine RNAiMAX Transfection Reagent. Cells were harvested 48 hours post-transfection followed by cDNA synthesis and gene expression profiling using TaqMan Gene Expression Cells-to-CT Kit and TaqMan Real-Time PCR assays. TaqMan qPCR assays were performed in triplicates and remaining expression was normalized to a scrambled (Scr siRNA) negative control (Scr) siRNA transfected sample.
Cells maintain high cell viability post siRNA transfection
Transfection reagents alone or in combination with siRNAs may lead to a nonspecific decrease in cell viability. This may impact interpretation of knockdown results. Five different siRNA against seven different lncRNA were transfected into the indicated cells. No significant decrease in viability was detected.
Figure 2. 25 nM of each siRNA was used to transfect 1X104 HeLa or HEK293T cells in 96-well plate format using Lipofectamine RNAiMAX transfection reagent. Forty-eight hours post transfection and prior to performing TaqMan real-time PCR assay cell viability was analyzed using the PrestoBlue Cell Viability assay. Percent cell viability was normalized to samples treated with Lipofectamine RNAiMAX transfection reagent without any siRNA.
Case study: Robust knockdown of MALAT-1, a nuclear localized lncRNA, with multiple siRNAs designed using the Ambion Silencer Select pipeline
Metastasis-associated lung adenocarcinoma transcript 1 (MALAT-1) is a lncRNA expressed from chromosome 11 and is known to be mis-regulated in several human carcinomas. MALAT-1 lncRNA is expressed in the nucleus, and its overexpression is implicated in the development and progression of numerous malignant cancers.
MALAT-1 expression was confirmed 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).
siRNAs designed against MALAT-1 were transfected into HeLa and A549 cells using Lipofectamine RNAiMAX Transfection Reagent. Knockdown was evaluated by real time PCR. 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 3).
Figure 3. Robust knockdown of nuclear localized MALAT-1 ncRNA in HeLa cells by multiple Silencer Select siRNAs and confirmation of results by northern analysis.
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