RNA quantification

Get tips on using TRIzol Reagent to perform RNA isolation / purification Cells - primary human aortic smooth muscle cells

Get tips on using TRIzol Reagent to perform RNA isolation / purification Cells - primary bovine pulmonary artery endothelial cells

Get tips on using TRIzol Reagent to perform RNA isolation / purification Cells - primary bovine coronary artery endothelial cells

Get tips on using TRIzol™ LS Reagent to perform RNA isolation / purification Bacteria - Gram positive Streptococcus pyogenes

Get tips on using TRIzol™ LS Reagent to perform RNA isolation / purification Bacteria - Gram positive Listeria monocytogens

Get tips on using TRI Reagent™ Solution to perform RNA isolation / purification Bacteria - Gram negative Salmonella typhi

Get tips on using TRI Reagent® Sigma to perform RNA isolation / purification Bacteria - Gram negative Helicobacter pylori

Get tips on using TRI Reagent® Sigma to perform RNA isolation / purification Cells - primary human cardiac fibroblasts

Get tips on using TRI Reagent® Sigma to perform RNA isolation / purification Cells - immortalized Mono-Mac-6

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.