cell-cycle-assay-human-hela

Get tips on using CD11b Antibody, anti-human/mouse, FITC to perform Flow cytometry Anti-bodies Human - CD11b

Get tips on using APC anti-human CD90 (Thy1) Antibody to perform Flow cytometry Anti-bodies Human - CD90

Get tips on using APC-H7 Mouse Anti-Human CD44 to perform Flow cytometry Anti-bodies Human - CD44

Get tips on using CD133/2 Antibody, anti-human, APC to perform Flow cytometry Anti-bodies Human - CD133

Get tips on using CD133/2 Antibody, anti-human, PE to perform Flow cytometry Anti-bodies Human - CD133

Get tips on using Human PAI1 ELISA Kit (SERPINE1) (ab184863) to perform ELISA Human - Serpin E1/PAI-1

Get tips on using Human Angiopoietin-like 3 DuoSet ELISA to perform ELISA Human - Angiopoietin-Like 3 (AngptL3)

Get tips on using Human Genome CGH Microarray 244A Supplemental to perform Microarray Comperative genomic hybridization - Human STUMP

Get tips on using Human Genome CGH Microarray Kit 244A to perform Microarray Comperative genomic hybridization - Human HepG2

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.