DNA methylation profiling Gene specific profiling Mouse muscle stem cells

Get tips on using Human/Mouse NLRP3/NALP3 Antibody to perform Western blotting NLRP3

Get tips on using Human/Mouse/Rat SOX2 Antibody to perform Western blotting SOX2

Get tips on using PCNA (PC10) Mouse mAb #2586 to perform Western blotting PCNA

Get tips on using Mouse Rat Estradiol ELISA Kit to perform ELISA Rat - Estradiol

Get tips on using Mouse GDNF ELISA Kit (ab171178) to perform ELISA Human - GDNF

Get tips on using Human/Mouse BDNF DuoSet ELISA to perform ELISA Human - BDNF

Get tips on using ApoAlert™ DNA Fragmentation Assay Kit to perform TUNEL assay cell type - Islets of langerhans (Beta cells)

Get tips on using ApoAlert™ DNA Fragmentation Assay Kit to perform TUNEL assay cell type - HeLa cells human cervical cancer

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 Human/Mouse/Rat/Canine ALCAM/CD166 Antibody to perform Immunohistochemistry Mouse - CD166 / ALCAM