Flowcytometry TLR2 (CD282) Mouse / IgG1, kappa

Get tips on using NOTCH1/activated Notch1 Antibody to perform Immunohistochemistry Mouse - Notch1

Get tips on using Lysozyme EC 3.2.1.17 (Concentrate) to perform Immunohistochemistry Mouse - Lysozyme

Get tips on using ChromaFlash Chromatin Extraction Kit to perform ChIP Mouse - Osteoblasts

Get tips on using EpiTect ChIP OneDay Kit to perform ChIP Mouse - MIN6

Get tips on using EpiTect ChIP qPCR Assays to perform ChIP Mouse - NIH3T3

Get tips on using HSP70 ELISA Kit (ab133060) to perform ELISA Mouse - HSP70

Get tips on using Muse® Cell Cycle Assay Kit to perform Cell cycle assay mouse - C2C12

Get tips on using Muse® Cell Cycle Assay Kit to perform Cell cycle assay mouse - L929

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 TRIzol Reagent to perform RNA isolation / purification Tissue - Mouse Muscle