Stem cell Differentiation media

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.

Get tips on using MitoSOX™ Red Mitochondrial Superoxide Indicator, for live-cell imaging to perform ROS assay cell type - PC-3 human prostate adenocarcinoma

Get tips on using CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) to perform

Get tips on using LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit to perform Live / Dead assay mammalian cells - mouse keratinocytes

Get tips on using Flp-In™ T-REx™ 293 Cell Line to perform Protein expression and purification Mammalian cells - HeLa ChaC1

Get tips on using EZViable™ Calcein AM Cell Viability Assay Kit (Fluorometric) to perform Live / Dead assay mammalian cells - rat brain microvascular endothelial cells

Get tips on using Flp-In™ T-REx™ 293 Cell Line to perform Protein expression and purification Mammalian cells - CAL-51 BRCA1