siRNA / miRNA gene silencing Human BCBL-1

Get tips on using Rab 7 siRNA (h) to perform siRNA / miRNA gene silencing Human - BEAS-2B RAB7A

Get tips on using Rab 5C siRNA (h) to perform siRNA / miRNA gene silencing Human - BEAS-2B RAB5C

Get tips on using AMPKα1/2 siRNA (h) to perform siRNA / miRNA gene silencing Human - HepG2 AMPKα1/α2

Get tips on using SMARTpool: siGENOME PAK4 siRNA to perform siRNA / miRNA gene silencing Human - OVCAR-3 PAK4

Get tips on using siRNA Junction plakoglobin (SASI_Hs01_00246520) to perform siRNA / miRNA gene silencing Human - BxPC-3 plakoglobin

Get tips on using NGFR p75 siRNA (h) to perform siRNA / miRNA gene silencing Human - HUVEC NGFR p75

Get tips on using AMPKα1/2 siRNA (h) to perform siRNA / miRNA gene silencing Human - HepG2 AMPKα1/α2

Get tips on using CREB-2 siRNA (h) to perform siRNA / miRNA gene silencing Human - HUVEC ATF4 Lipid

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 have 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 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 have 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 efficacy of transduction and shRNA on its target site.