Site Directed Mutagenesis (SDM) Human Point mutation HEK293

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.

A PCR reaction consists of the template DNA, two primers covering the amplification site, an enzyme, and buffers. However, non-specific product amplification and primer-dimer formation during set-up are major causes of PCR failure. Nevertheless, high-quality hot-start DNA polymerase and optimize reaction buffers will certainly lead to a successful PCR reaction

Get tips on using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003 to perform ChIP Human - SMMC-7721

Get tips on using AmpFLSTR™ Identifiler™ Direct PCR Amplification Kit to perform Cell line authentication Human iPSC cells derived from human dermal fibroblasts

Get tips on using Silencer® Select GLO-1 siRNA to perform siRNA / miRNA gene silencing Human - Primary Human Aortic Endothelial Cells GLO-1 Lipid

Get tips on using SIRT1 siRNA and shRNA Plasmids (h) to perform siRNA / miRNA gene silencing Human - PC3 (human prostate cancer cell line) SIRT1

Get tips on using In situ apoptosis detection to perform Apoptosis assay cell type - Human endometrial stromal cells

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

Get tips on using heat shock protein family A (Hsp70) member 5 to perform siRNA / miRNA gene silencing Human - PC3 (human prostate cancer cell line) HSPA5 (GRP78)

Get tips on using Stealth siRNA_GATA2 to perform siRNA / miRNA gene silencing Human - LAD2 GATA2