Knockdown of BDNF-AS1 in hNDF cells by Lincode SMARTpool and four siRNAs. All siRNAs were used at 25 nM, normalized and remaining lncRNA transcripts were detected with Solaris (sold under the Thermo Scientific brand) qPCR lncRNA Expression Assays and normalized to Non-targeting Control siRNA. Viability was assessed by resazurin assay.
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.
Lincode siRNA effectively knocks down BDNF-AS1 lncRNA, but not the protein-coding transcript BDNF that is antisense to the lncRNA target. This indicates strand specificity of Lincode siRNAs and effectiveness of ON TARGETplus modifications. siRNAs were applied at the indicated concentrations to hNDF cells. BDNF-AS1 and BDNF transcripts were detected with Solaris qPCR Expression Assays and normalized to Non-targeting control siRNA. Viability was assessed by resazurin assay and normalized to Untreated.