crispr-mouse-deletion-raw-264-7-dcstamp

Get tips on using BrdU Cell Proliferation Assay to perform Cell cytotoxicity / Proliferation assay cell type - MCF-7

Get tips on using Cell Counting Kit-8 to perform Cell cytotoxicity / Proliferation assay cell type - MCF-7

Get tips on using SV Total RNA Isolation System to perform RNA isolation / purification Cells - immortalized MCF-7

Get tips on using PureLink™ RNA Mini Kit to perform RNA isolation / purification Cells - immortalized MCF-7

Get tips on using HyClone™ Dulbecco's Modified Eagles Medium to perform Mammalian cell culture media MCF-7

Get tips on using BD Cycletest™ Plus DNA Kit to perform Cell cycle assay human - MCF-7

Get tips on using Senescence Detection Kit - Merck to perform Reporter gene assay β-galactosidase substrates - MCF-7 human breast cancer

Get tips on using eBioscience™ Annexin V-FITC Apoptosis Detection Kit to perform Apoptosis assay cell type - Caspase 3/7

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.

Get tips on using lenti dCAS-VP64_Blast to perform CRISPR Human - Activation interferon-γ promoter