siRNA / RNAi /miRNA transfection Human Cal 27 cells

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jetPEI® DNA transfection, HTS application Product

Get tips on using jetPEI® DNA transfection, HTS application to perform DNA transfection Mammalian cells - Immortalized cell lines MDA-MB-231

Products Polyplus transfections jetPEI® DNA transfection, HTS application

mirVana® miRNA mimic Product

Get tips on using mirVana® miRNA mimic to perform RNA isolation / purification Cells - primary human endothelial cells

Products Thermo Fisher Scientific mirVana® miRNA mimic

shRNA gene silencing Human - HEK 293T CAPN5- (Calpains) cationic lipid based Experiment

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.

RNA shRNA gene silencing Human HEK 293T CAPN5- (Calpains) cationic lipid based

X-tremeGENE™ HP DNA Transfection Reagent Product

Get tips on using X-tremeGENE™ HP DNA Transfection Reagent to perform DNA transfection Mammalian cells - Immortalized cell lines HeLa