siRNA / RNAi /miRNA transfection Human Cells A549

Get tips on using Stealth siRNA_GATA2 to perform siRNA / miRNA gene silencing Human - LAD2 GATA2

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.

Get tips on using TRIzol Reagent to perform RNA isolation / purification Cells - immortalized A549

Get tips on using Fenozol to perform siRNA / miRNA gene silencing Human - BOSC23

Get tips on using PICK1 siRNA to perform siRNA / miRNA gene silencing Rat - Astrocytes PICK1

Get tips on using Fabp5 siRNA to perform siRNA / miRNA gene silencing Rat - PC12 Fabp5

Get tips on using Vps13a siRNA to perform siRNA / miRNA gene silencing Rat - PC12 chorein

Get tips on using Cnp siRNA to perform siRNA / miRNA gene silencing Mouse - BV2 CNPase

Get tips on using Wwtr1 siRNA to perform siRNA / miRNA gene silencing Mouse - C2C12 Taz

Get tips on using Bambi siRNA to perform siRNA / miRNA gene silencing Mouse - C2C12 BAMBI