Live / Dead assay bacteria

Get tips on using Carboxy-H2DCFDA (general oxidative stress indicator) to perform ROS assay cell type - PLHC-1, SK-HEP-1, Hep3b, HepG2 human hepatocellular carcinoma

Get tips on using ROS-ID® Total ROS/Superoxide detection kit to perform ROS assay cell type - A549 human adenocarcinomic human alveolar basal epithelial cells

Get tips on using ApopTag® Fluorescein In Situ Apoptosis Detection Kit to perform TUNEL assay cell type - A549, NCI-H460, H1299 human lung cancer cells

Get tips on using FragEL™ DNA Fragmentation Detection Kit, Colorimetric - TdT Enzyme to perform TUNEL assay cell type - A127, U87MG, U251MG, T98G human glioblastoma cells

Get tips on using ApopTag® Peroxidase In Situ Apoptosis Detection Kit to perform TUNEL assay cell type - HNSCC Detroit 562 human head and neck tumor cells

Get tips on using ROS-ID® Total ROS/Superoxide detection kit to perform ROS assay cell type - PLHC-1, SK-HEP-1, Hep3b, HepG2 human hepatocellular carcinoma

Get tips on using AllStars Hs Cell Death siRNA to perform siRNA / miRNA gene silencing Human - U2OS KRAS

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.