DNA transfection Mammalian cells Immortalized cell lines

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

Get tips on using In Situ Cell Death Detection Kit, Fluorescein to perform TUNEL assay cell type - SKOV3, Caov3 human ovarian cancer

Get tips on using in situ Cell Death Detection Kit, POD to perform TUNEL assay cell type - PANC-1 human pancriatic cancer

Get tips on using In Situ Cell Death Detection Kit, Fluorescein to perform TUNEL assay cell type - PC-3 human prostate cancer

Get tips on using In Situ Cell Death Detection Kit, Fluorescein to perform TUNEL assay cell type - 3T3 L1 mouse adipose tissue

Get tips on using In Situ Cell Death Detection Kit, Fluorescein to perform TUNEL assay cell type - SK-MEL-2 human melanoma

Get tips on using StemSpan™ SFEM to perform Stem cell Differentiation media hiPSCs differentiation into mesodermal lineage cells

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 Cell Counting Kit-8 to perform RNA quantification Coloremetric

Get tips on using in situ Cell Death Detection Kit, POD to perform TUNEL assay cell type - A549, NCI-H460, H1299 human alveolar carcinoma