siRNA / miRNA gene silencing Human HCT-116

Get tips on using stealth siRNA GIRK1/KCNJ3 to perform siRNA / miRNA gene silencing Human - MDA-MB-453 GIRK1/KCNJ3

Get tips on using stealth siRNA GIRK1/KCNJ3 to perform siRNA / miRNA gene silencing Human - MDA-MB-231 GIRK1/KCNJ3

Get tips on using Dlx-2 siRNA (h) to perform siRNA / miRNA gene silencing Human - MDA-MB-231 DLX-2

Get tips on using Bcl-2 siRNA (h) to perform siRNA / miRNA gene silencing Human - MDA-MB-231 bcl-2

Get tips on using PI 3-kinase p100 siRNA (h) to perform siRNA / miRNA gene silencing Human - BEAS-2B PIK3C3

Get tips on using mirVana® miRNA mimic to perform siRNA / miRNA gene silencing Human - Primary Endometrial Stromal Cells hsa-miR-542-3p

Get tips on using OCT4-PG1 siRNA to perform siRNA / miRNA gene silencing Human - hES cell line H1 (WA01) OCT4-PG1

Get tips on using siRNA MAPKAPK-2 to perform siRNA / miRNA gene silencing Human - U937 MK2 (MAPK Kinase 2) Viral vectors

Get tips on using siRNA MAPKAPK-2 to perform siRNA / miRNA gene silencing Human - Jurkat MK2 (MAPK Kinase 2) Viral vectors

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.