Detection of effective lncRNA knockdown following application of Lincode siRNA reagents.
All siRNAs used at 25 nM. Detection of remaining lncRNA by corresponding Solaris (sold under the Thermo Scientific brand) qPCR lncRNA expression assay.
(A) Knockdown of CDKN2B-AS1 in HeLa cells; normalized to Lincode Non-targeting siRNA #4 (D-001320-04)
(B) Knockdown of BDNF-AS1 in hNDF cells; normalized to Lincode Non-targeting siRNA #2 (D-001320-02)
Robust qPCR assays are necessary for accurate determination of lncRNA expression levels.
It is of vital importance to determine lncRNA expression in your cells prior to knockdown experiments. lncRNA expression can be expected to vary much more than is typically observed from protein-coding genes like Cyclophilin B (PPIB).
lncRNAs demonstrate lower and more variable expression than protein-coding genes. It is therefore highly recommend to determine lncRNA expression levels in your cells prior to knockdown experiments. Solaris qPCR expression assays are designed for sensitive detection of lncRNA and demonstrate high sensitivity and reproducibility.
50 ng of total RNA was converted into cDNA using the Maxima First Strand cDNA Synthesis Kit (#K1641). Gene expression levels were evaluated with Solaris qPCR assays. Cyclophilin B (PPIB) was used as a reference for typical mRNA expression. Standard RT-PCR cycling conditions resulted in initial Cq values ranging from 22 (PPIB in 293T cells) to 36 (BDNF-AS1 in HeLa cells). MLK7-AS1 lncRNA expression was not detectable in any of the cell lines tested. Cell lines include HeLa, HEK293T, Human Neonatal Dermal Fibroblast (hNDF).
Lincode siRNAs are modified with a proprietary dual-strand modification that improves siRNA specificity
Additionally, off-targets are reduced due to:
- Inactivation of sense strand activity; driving preferential loading of the antisense strand into RISC
- Novel antisense seed region modification for disruption of microRNA-like off-targets
Genomic context and Lincode siRNA targeting of BDNF-AS1 lncRNA.
BDNF-AS1 lncRNA is anti-sense to BDNF protein-coding RNA. Direction of transcription from genomic DNA is indicated by arrows, exons are indicated by rectangles. Position of the Lincode siRNA target is indicated by double green lines.
Lincode siRNA modifications prevent sense strand activity.
qPCR results indicate of a protein coding transcript (BDNF) with Lincode siRNA targeting a lncRNA on its antisense strand (BDNF-AS1), indicating strand specificity of lncRNA siRNA design and effectiveness of ON-TARGETplus modifications.
Genomewide expression analysis demonstrates improved specificity of Lincode siRNA.
To detect subtle phenotypic changes that may arise from lncRNA knockdown, it is essential to incorporate strategies to prevent the off-targeting of protein-coding genes. Lincode siRNA reagents are synthesized with proprietary dual-strand modifications known as the ON-TARGETplus modification pattern which has been proven to reduce off-targets arising from microRNA-like activity of the antisense seed region of the siRNA.
Two different siRNAs targeting the same lncRNA were synthesized with modifications that (a) block the sense strand only or (b) the ON-TARGETplus modification pattern which blocks the sense strand and includes an antisense strand seed region modification. While the target lncRNA was effectively silenced by all four siRNAs (arrows), the siRNAs synthesized with the ON-TARGETplus modifications demonstrated greatly reduced off-targets.
Agilent™ G3 Human Gene Expression Microarray™ HeLa 12K, harvest 24 hrs post transfection, 100 nM siRNA, Analysis: >2 fold down regulated, pval <0.05, and non-siRNA-specific effects filtered out.