siRNA / miRNA gene silencing Human HeLa EPAS-1

Get tips on using ON-TARGET plus Mouse Becn1 (56208) siRNA - SMARTpool to perform siRNA / miRNA gene silencing Mouse - 4T1 BECN-1

Get tips on using B2M siRNA to perform siRNA / miRNA gene silencing Human - hES cell line H1 (WA01) B2M

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.

Get tips on using pcDNA™3.1 (+) Mammalian Expression Vector to perform siRNA / miRNA gene silencing Human - U251 cofilin-1 (CFL1)

Get tips on using N-WASP siRNA (h) to perform siRNA / miRNA gene silencing Human - T47-D N-WASP

Get tips on using VEGF-D siRNA (h) to perform siRNA / miRNA gene silencing Human - Caki-2 VEGF-D

Get tips on using IL-8 siRNA (h) to perform siRNA / miRNA gene silencing Human - HUVEC IL-8 Lipid

Cell cycle can be challenging due to difference introduced by sample handling, timing, and difference within the sample. Downstream instriuments to analyse cell cycle (Multicolor flow cytometry and multicolor imaging) can answer these challenges. Relevant markers can be combined with cell phenotyping markers to look at events within subpopulations of cells.

Get tips on using Rock-2 siRNA and shRNA Plasmids (h) to perform siRNA / miRNA gene silencing Human - HT-1376 ROCK2