rna-isolation-purification-cells-primary-human-carotid-artery-endothelial-cells

Get tips on using CelLytic™ M to perform Protein isolation Mammalian cells - HaCaT

Get tips on using CelLytic™ M to perform Protein isolation Mammalian cells - HepG2

Get tips on using CelLytic™ M to perform Protein isolation Mammalian cells - Huh7

Get tips on using CelLytic™ M to perform Protein isolation Mammalian cells - HEK293T

Get tips on using Live and Dead Cell Assay (Abcam) to perform Live / Dead assay mammalian cells - human Mesenchymal stem cells

Get tips on using pLEXSY-hyg-2-hAm to perform Protein Expression Eukaryotic cells - Iranian lizard Leishmania cells recombinant human amelogenin

Get tips on using Pierce™ Coomassie (Bradford) Protein Assay Kit to perform Protein quantification Mammalian cells - Human pluripotent stem cells

Get tips on using Ambion™ RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE to perform DNA isolation / purification Bacteria - Gram positive Lactobacillus

Get tips on using Cell Death Detection ELISA to perform TUNEL assay cell type - Rat pulmonary arterial smooth muscle 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.