siRNA / miRNA gene silencing Mouse C2C12

Get tips on using Rn_Tlr3_1 FlexiTube siRNA to perform siRNA / miRNA gene silencing Mouse - Neuro 2a TLR3

Get tips on using Atf4 siRNA(m) to perform siRNA / miRNA gene silencing Mouse - 3T3-L1 ATF4

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.

Site-directed mutagenesis (SDM) can be challenging, particularly during detection/confirmation of (SDM) in colonies by sequencing or PCR techniques. This common issue in SDM is heavily relying on designing of mutagenic primer pairs. The best solution is to design the mutagenic primers that have extended 3'-ends/3'-overhang. This would provide the annealing region between the mutagenic primer pair is essentially shorter. and hence ensure a lower annealing temperature for the primer pair along with a higher chance of annealing to the template.

Site-directed mutagenesis (SDM) can be challenging, particularly during detection/confirmation of (SDM) in colonies by sequencing or PCR techniques. This common issue in SDM is heavily relying on designing of mutagenic primer pairs. The best solution is to design the mutagenic primers that have extended 3'-ends/3'-overhang. This would provide the annealing region between the mutagenic primer pair is essentially shorter. and hence ensure a lower annealing temperature for the primer pair along with a higher chance of annealing to the template.

Get tips on using Stealth siRNA(m)_Atg5 to perform siRNA / miRNA gene silencing Mouse - MLO‐Y4 Atg5

Get tips on using IRF-1 siRNA (m) to perform siRNA / miRNA gene silencing Mouse - B16-F10 IRF1

Get tips on using HIF-1α siRNA (m) to perform siRNA / miRNA gene silencing Mouse - AtT20 Hif-1alpha

Get tips on using MEK-3 siRNA (m) to perform siRNA / miRNA gene silencing Mouse - BMDMs MEK-3

Get tips on using PU.1 siRNA (m) to perform siRNA / miRNA gene silencing Mouse - RAW264.7 PU.1