siRNA / RNAi /miRNA transfection Rat AR42J

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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.

RNA shRNA gene silencing Rat WKY Salusin-β

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.

RNA shRNA gene silencing Rat H9c2 salusin-β

Get tips on using Net siRNA (m) to perform siRNA / miRNA gene silencing Mouse - FL83B Elk3/Net

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Get tips on using ATG5 siRNA (m) to perform siRNA / miRNA gene silencing Mouse - NIH-3T3 Atg5

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Get tips on using p130 siRNA (m) to perform siRNA / miRNA gene silencing Mouse - mESC Rbl2/p130

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

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Get tips on using TLR4 siRNA (m) to perform siRNA / miRNA gene silencing Mouse - Neuro 2a TLR4

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Get tips on using C23 siRNA (m) to perform siRNA / miRNA gene silencing Mouse - Neuro 2a C23

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Get tips on using ANT2 siRNA (m) to perform siRNA / miRNA gene silencing Mouse - 3T3-L1 ANT2

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Get tips on using Stealth Select_PTBP1 siRNA to perform siRNA / miRNA gene silencing Human - HCT-116 PTBP1

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