siRNA / miRNA gene silencing Human Primary Endometrial Stromal Cells hsa-miR-542-3p

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.

Get tips on using FragEL™ DNA Fragmentation Detection Kit, Colorimetric - TdT Enzyme to perform Apoptosis assay cell type - Human endometrial stromal cells

Get tips on using ON-TARGETplus Human LRRC8A (56262) siRNA - SMARTpool to perform siRNA / miRNA gene silencing Human - A2780 LRRC8A

Get tips on using ON-TARGETplus Human SMU1 (55234) siRNA - Individual to perform siRNA / miRNA gene silencing Human - HeLa SMU1

Get tips on using ON-TARGETplus Human PLAU (5328) siRNA - SMARTpool to perform siRNA / miRNA gene silencing Human - DU145 PLAU

Get tips on using ON-TARGETplus human ATG16L1 siRNA to perform siRNA / miRNA gene silencing Human - SHSY5Y ATG16L1

Get tips on using ON-TARGETplus Human TET1 siRNA to perform siRNA / miRNA gene silencing Human - A172 TET1

Get tips on using ON-TARGETplus Human PIK3CB (5291) siRNA - SMARTpool to perform siRNA / miRNA gene silencing Human - A498 PIK3CB

Get tips on using ON-TARGETplus Human PIK3CA (5290) siRNA - SMARTpool to perform siRNA / miRNA gene silencing Human - A498 PIK3CA

Get tips on using ON-TARGETplus Human PIK3CD (5293) siRNA - SMARTpool to perform siRNA / miRNA gene silencing Human - A498 PIK3CD