ChIP H3K27me3 Human Mouse

Get tips on using Human Decorin DuoSet ELISA to perform ELISA Human - Decorin

Get tips on using Human FGF-10 Antibody to perform Immunohistochemistry Human - FGF-10

Get tips on using Human Dkk-1 ELISA to perform ELISA Human - Dkk-1

Get tips on using siGENOME Human MINK1 siRNA to perform siRNA / miRNA gene silencing Human - RMS MINK

Get tips on using siGENOME Human MAP4K2 siRNA to perform siRNA / miRNA gene silencing Human - RMS MAP4K2

Get tips on using esiRNA human PTPN3 (esiRNA1) to perform siRNA / miRNA gene silencing Human - A2780 PTPN3

Get tips on using siGENOME Human PAK1 siRNA to perform siRNA / miRNA gene silencing Human - HeLa PAK1

Get tips on using ON-TARGETplus SMARTpool - Human to perform siRNA / miRNA gene silencing Human - U2OS DKC1

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.