Get tips on using Rat sE-Selectin ELISA Kit (sCD62E) (ab171334) to perform ELISA Rat - sE-Selectin
Get tips on using Rat/Mouse Cytochrome c Quantikine ELISA Kit to perform ELISA Rat - Cytochrome c
Get tips on using Rat BMP-2 PicoKine™ ELISA Kit to perform ELISA Rat - BMP-2
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 Rat C-Reactive Protein/CRP DuoSet ELISA to perform ELISA Rat - C-Reactive Protein/CRP
Get tips on using HyClone Dulbecco's Modified Eagle Medium (DMEM)/ F12 1:1: Liquid to perform Stem cell Differentiation media hDPSCs differentiation into adipogenic cells
Get tips on using HyClone Dulbecco's Modified Eagle Medium (DMEM)/ F12 1:1: Liquid to perform Stem cell Differentiation media hDPSCs differentiation into osteogenic cells
Get tips on using PE Rat Anti-Mouse CD103 to perform Flow cytometry Anti-bodies Mouse - CD103
Get tips on using PE Rat Anti-Mouse CD25 to perform Flow cytometry Anti-bodies Mouse - CD25
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