rna-isolation-purification-cells-primary-mouse-cortical-neurons

Get tips on using FuGENE® 6 Transfection Reagent to perform DNA transfection Mammalian cells - Primary cells Rat pulmonary artery smooth muscle cell (pPASMC)

Get tips on using GenJet™ In Vitro DNA Transfection Reagent to perform DNA transfection Mammalian cells - Primary cells Human lung fibroblasts (HLF)

Get tips on using Mouse TNFSF11/RANKL PicoKine™ ELISA Kit to perform ELISA Mouse - RANK L

Get tips on using PE anti-mouse CD146 Antibody to perform Flow cytometry Anti-bodies Mouse - CD146/MCAM

Get tips on using InVivoMAb anti-mouse CD16/CD32 to perform Flow cytometry Anti-bodies Mouse - CD16/CD32

Get tips on using Tlr4 Mouse siRNA Oligo Duplex to perform siRNA / miRNA gene silencing Mouse - BV2 TLR4

Get tips on using ON-TARGETplus Mouse Lgals3 siRNA to perform siRNA / miRNA gene silencing Mouse - BV2 LGAL3S3

Get tips on using ON-TARGETplus Mouse Samhd1 siRNA to perform siRNA / miRNA gene silencing Mouse - BMDMs SAMHD1

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.

Get tips on using PE Hamster Anti-Mouse CD279 to perform Flow cytometry Anti-bodies Mouse - CD279/PD-1