siRNA / miRNA gene silencing Human Aortic smooth muscle cell

Get tips on using Ccr2 siRNA to perform siRNA / miRNA gene silencing Rat - Glial cells CCR2

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.

When extracting nucleic acids from cell cultures, thorough homogenization of cells via vortexing in lysis buffer is very necessary. Choose the best RNA isolation method keeping in mind the downstream applications, generally, column-based isolations result in clean and concentrated RNA samples. Downstream applications like sequencing and cDNA synthesis require high-quality RNA, always treat the samples with DNases and check their integrity by running a gel.

When extracting nucleic acids from cell cultures, thorough homogenization of cells via vortexing in lysis buffer is very necessary. Choose the best RNA isolation method keeping in mind the downstream applications, generally, column-based isolations result in clean and concentrated RNA samples. Downstream applications like sequencing and cDNA synthesis require high-quality RNA, always treat the samples with DNases and check their integrity by running a gel.

Get tips on using FGD4 siRNAs (FlexiTube) to perform siRNA / miRNA gene silencing Human - LNCap FGD4

Get tips on using Timp2 siRNA to perform siRNA / miRNA gene silencing Mouse - BV2 TIMP-2

Get tips on using NFE2L2 siRNA to perform siRNA / miRNA gene silencing Mouse - B16-F10 Nrf2

Get tips on using Kdm2b siRNA to perform siRNA / miRNA gene silencing Mouse - 3T3-L1 Fbxl10

Get tips on using Stk11 siRNA to perform siRNA / miRNA gene silencing Mouse - 3T3-L1 Stk11

Get tips on using Ccl2 siRNA to perform siRNA / miRNA gene silencing Rat - Neuronal cells MCP-1