Get tips on using ON-TARGETplus Rat Atf4 (79255) siRNA - SMARTpool to perform siRNA / miRNA gene silencing Rat - PC12 Atf4
Get tips on using ON-TARGETplus Rat Dcp1a (361109) siRNA - SMARTpool to perform siRNA / miRNA gene silencing Rat - PC12 Dcp1a
Get tips on using ON-TARGETplus Rat Lsm1 (364624) siRNA - SMARTpool to perform siRNA / miRNA gene silencing Rat - PC12 Lsm1
Get tips on using siGENOME Rat Acvr1c (245921) siRNA - SMARTpool to perform siRNA / miRNA gene silencing Rat - INS-1 Alk7/Acvr1c
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.
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 Ccl2 siRNA to perform siRNA / miRNA gene silencing Rat - Neuronal cells MCP-1
Get tips on using ON-TARGETplus Rat Smad2 (29357) siRNA - SMARTpool to perform siRNA / miRNA gene silencing Rat - IEC-6 Smad2
Get tips on using ON-TARGETplus Rat Smad3 (25631) siRNA - SMARTpool to perform siRNA / miRNA gene silencing Rat - IEC-6 Smad3
Get tips on using ON-TARGETplus Rat Hspa5 (25617) siRNA - SMARTpool to perform siRNA / miRNA gene silencing Rat - AR42J Grp78/Hspa5
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