Wound healing assay cell type rat

Get tips on using eBioscience™ Annexin V-FITC Apoptosis Detection Kit to perform Apoptosis assay cell type - MCF10A

Get tips on using Phospho-PI3 Kinase p85 (Tyr458)/p55 (Tyr199) Antibody to perform Autophagy assay cell type - HepG2

Get tips on using Anti-PI 3 Kinase p85 alpha antibody [EP380Y] to perform Autophagy assay cell type - HepG2

Get tips on using ApopTag® Peroxidase In Situ Apoptosis Detection Kit to perform Apoptosis assay cell type - HEK293

Get tips on using FITC Annexin V Apoptosis Detection Kit I (RUO) to perform Apoptosis assay cell type - HUVEC

Get tips on using FITC Annexin V Apoptosis Detection Kit I (RUO) to perform Apoptosis assay cell type - L02

Get tips on using FITC Annexin V Apoptosis Detection Kit I (RUO) to perform Apoptosis assay cell type - JJ012

Get tips on using mRFP-green fluorescent protein (GFP)-LC3 adenoviral vector to perform Autophagy assay cell type - Macrophages

Get tips on using Anti-Collagen, Type VII to perform Immunohistochemistry Collagen Type VII - Rabbit Human -NA-

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.