siRNA / miRNA gene silencing Human SUIT-2

Get tips on using ON-TARGETplus Human CDK9 (1025) siRNA - SMARTpool to perform siRNA / miRNA gene silencing Human - A549 CDK9

Get tips on using ON-TARGETplus Human IRF3 (3661) siRNA - SMARTpool to perform siRNA / miRNA gene silencing Human - A549 IRF3

Get tips on using ON-TARGETplus Human MET (4233) siRNA - SMARTpool to perform siRNA / miRNA gene silencing Human - A431 MET

Get tips on using ON-TARGETplus Human PCSK6 (5046) siRNA - Individual to perform siRNA / miRNA gene silencing Human - A253 PACE4

Get tips on using ON-TARGETplus Human FURIN (5045) siRNA - SMARTpool to perform siRNA / miRNA gene silencing Human - A253 Furin

Get tips on using ON-TARGETplus Human ITGB4 (3691) siRNA - SMARTpool to perform siRNA / miRNA gene silencing Human - CAL-27 ITGB5

Get tips on using ON-TARGETplus Human DPAGT1 (1798) siRNA - SMARTpool to perform siRNA / miRNA gene silencing Human - CAL-27 DPAGT1

Get tips on using ON-TARGETplus Human CTHRC1 (115908) siRNA - SMARTpool to perform siRNA / miRNA gene silencing Human - CAL-27 CTHRC1

Short hairpin or small hairpin RNA (shRNA) is artificial RNA, which has a hairpin loop structure, and uses inherent microRNA (miRNA) machinery to silence target gene expression. This is called RNA interference (RNAi). These can be delivered via plasmids or viral/bacterial vectors. Challenges in shRNA-mediated gene silencing include 1. Off-target silencing, 2. Packaging shRNA encoding lentivirus, and 3. Stable transduction in cells. RNAi has been designed to have anywhere from 19-27 bs, but the most effective design has 19 bp. In case commercial shRNAs are not available, potential target sites can be chosen within exon, 5’- or 3’ UTR, depending on which splice variants of the gene are desired. One should use the latest algorithms and choose at least two different sequences, targeting different regions, in order to have confidence in overcoming off-target effects. A BLAST search after selecting potential design will eliminate potential off-target sequences. For the second challenge, sequencing the vector using primers for either strand (50-100 bp upstream) is suggested, along with using enzymatic digestion on agarose gel for the vector. Next, once the shRNA-containing vector is packaged in a virus, it is important to check the viral titer before transduction. Finally, using a marker in the lentiviral vector (fluorescent protein or antibiotic resistance), along with qPCR for target gene expression can help in determining the efficacy of transduction and shRNA on its target site.

Short hairpin or small hairpin RNA (shRNA) is artificial RNA, which has a hairpin loop structure, and uses inherent microRNA (miRNA) machinery to silence target gene expression. This is called RNA interference (RNAi). These can be delivered via plasmids or viral/bacterial vectors. Challenges in shRNA-mediated gene silencing include 1. Off-target silencing, 2. Packaging shRNA encoding lentivirus, and 3. Stable transduction in cells. RNAi has been designed to have anywhere from 19-27 bs, but the most effective design has 19 bp. In case commercial shRNAs are not available, potential target sites can be chosen within exon, 5’- or 3’ UTR, depending on which splice variants of the gene are desired. One should use the latest algorithms and choose at least two different sequences, targeting different regions, in order to have confidence in overcoming off-target effects. A BLAST search after selecting potential design will eliminate potential off-target sequences. For the second challenge, sequencing the vector using primers for either strand (50-100 bp upstream) is suggested, along with using enzymatic digestion on agarose gel for the vector. Next, once the shRNA-containing vector is packaged in a virus, it is important to check the viral titer before transduction. Finally, using a marker in the lentiviral vector (fluorescent protein or antibiotic resistance), along with qPCR for target gene expression can help in determining the efficacy of transduction and shRNA on its target site.