Protein expression and purification Bacteria

Get tips on using TRIzol Reagent to perform RNA isolation / purification Bacteria - Gram positive Clostridium tetani

Get tips on using TRIzol Reagent to perform RNA isolation / purification Bacteria - Gram negative Vibro parahaemolyticus

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

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

Get tips on using NucleoSpin® Plasmid to perform DNA isolation / purification Bacteria - Gram negative E.coli

Get tips on using NucleoSpin® Soil to perform DNA isolation / purification Bacteria - Gram positive Lactobacillus

Get tips on using MICROBExpress™ Bacterial mRNA Enrichment Kit to perform RNA isolation / purification Bacteria - Gram positive Staphylococcus aureus

Get tips on using MICROBExpress™ Bacterial mRNA Enrichment Kit to perform RNA isolation / purification Bacteria - Gram negative Pseudomonas aeruginosa

Get tips on using EMBacY#5 to perform Protein Expression Eukaryotic cells - Hi5 Soluble G protein (Hendra Virus)

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.