rna-isolation-purification-cells-immortalized-mda-mb-361

Get tips on using QIAprep Spin Miniprep Kit to perform Plasmid Isolation Pseudomonas aeruginosa

Get tips on using QIAGEN Large-Construct Kit to perform Plasmid Isolation Pseudomonas aeruginosa

Get tips on using QIAprep Spin Miniprep Kit to perform Plasmid Isolation Shigella flexneri

Get tips on using CompactPrep Plasmid Midi Kit to perform Plasmid Isolation Salmonella enterica

Get tips on using QIAprep Spin Miniprep Kit to perform Plasmid Isolation Lactococcus lactis

Get tips on using GeneJET Plasmid Miniprep Kit to perform Plasmid Isolation Lactobacillus brevis

Get tips on using GeneJET Plasmid Miniprep Kit to perform Plasmid Isolation Bacillus smithii

Get tips on using QIAprep Spin Miniprep Kit to perform Plasmid Isolation E. coli

Get tips on using QIAGEN Large-Construct Kit to perform Plasmid Isolation DH10Bac (Bacmid)

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.