Reporter gene assay β-lactamase substrates

Get tips on using MTT Cell Proliferation Assay (ATCC® 30-1010K™) to perform Cell cytotoxicity / Proliferation assay cell type - MCF-7

Get tips on using Viability/Cytotoxicity Assay Kit for Animal Live & Dead Cells to perform Live / Dead assay mammalian cells - human fibroblast tissue

Get tips on using Viability/Cytotoxicity Assay Kit for Animal Live & Dead Cells to perform Live / Dead assay mammalian cells - L29 mouse fibroblast

Get tips on using Pierce™ Coomassie (Bradford) Protein Assay Kit to perform Protein quantification Tissue - mouse liver

Get tips on using Pierce™ Coomassie (Bradford) Protein Assay Kit to perform Protein quantification Tissue - mouse kidney

Get tips on using Pierce™ Coomassie (Bradford) Protein Assay Kit to perform Protein quantification Tissue - mouse brain

Get tips on using Pierce™ Coomassie (Bradford) Protein Assay Kit to perform Protein quantification Mammalian cells - RAW264.7

Get tips on using DCFDA / H2DCFDA - Cellular Reactive Oxygen Species Detection Assay Kit to perform ROS assay cell type - L-02 human fetal hepatocyte

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 Human ANGPTL3 (highly sensitive) Assay Kit (27750 ) to perform ELISA Human - Angiopoietin-Like 3 (AngptL3)