siRNA / miRNA gene silencing Rat Retinal stem cells

Get tips on using STEAP1 siRNA to perform siRNA / miRNA gene silencing Human - LNCap STEAP1

Get tips on using TruSeq Stranded mRNA to perform RNA sequencing Rat - Retinal ganglion cells (RGCs)

Get tips on using YAP siRNA (h) to perform siRNA / miRNA gene silencing Human - OV2008 Yap Gene

Get tips on using SiRNA silencing human Eph receptor B4, Id: s243 to perform siRNA / miRNA gene silencing Human - HNSCC cell line Eph receptor B4 Polymer / Lipid

Get tips on using SiRNA silencing human Eph receptor B4, Id: 533 to perform siRNA / miRNA gene silencing Human - HNSCC cell line Eph receptor B4 Polymer / Lipid

Get tips on using RAB8A siRNA to perform siRNA / miRNA gene silencing Human - HEK293 Rab8a

Get tips on using Dusp3 siRNA to perform siRNA / miRNA gene silencing Mouse - RAW264.7 Dusp3

Get tips on using Control siRNA-A to perform siRNA / miRNA gene silencing Human - OV2008 Yap Gene Lipofectamine

Get tips on using YAP siRNA (h) to perform siRNA / miRNA gene silencing Human - OV2008 Yap Gene Lipofectamine

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.