siRNA / miRNA gene silencing Human KLM-1

Get tips on using ON-TARGETplus Rat Pld2 (25097) siRNA - SMARTpool to perform siRNA / miRNA gene silencing Rat - RBL-2H3 Pld2

Get tips on using ON-TARGETplus Rat Arhgef12 (367072) siRNA - SMARTpool to perform siRNA / miRNA gene silencing Rat - MTLn3 Larg/Arhgef12

Get tips on using ON-TARGETplus Rat Arhgef1 (60323) siRNA - SMARTpool to perform siRNA / miRNA gene silencing Rat - MTLn3 p115RhoGEF/Arhgef1

Get tips on using ON-TARGETplus Rat Tmed10 (84599) siRNA - SMARTpool to perform siRNA / miRNA gene silencing Rat - NRK Tmp21/Tmed10

Get tips on using ON-TARGETplus Mouse Sphk1 (20698) siRNA - SMARTpool to perform siRNA / miRNA gene silencing Mouse - MC3T3-E1 SphK1

Get tips on using ON-TARGETplus Mouse Mprip (26936) siRNA - SMARTpool to perform siRNA / miRNA gene silencing Mouse - 3T3-SA Mprip

Get tips on using ON-TARGETplus Mouse Ddit4 (74747) siRNA - SMARTpool to perform siRNA / miRNA gene silencing Mouse - RGC-5 Ddit4

Get tips on using Flot1 Rat siRNA Oligo Duplex (Locus ID 64665) to perform siRNA / miRNA gene silencing Rat - NRCM Flot1

Get tips on using Flot2 Rat siRNA Oligo Duplex (Locus ID 83764) to perform siRNA / miRNA gene silencing Rat - NRCM Flot2

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.