siRNA / miRNA gene silencing Human Aortic smooth muscle cell

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.

As autophagy is a multi-step process which includes not just the formation of autophagosomes, but most importantly, flux through the entire system, including the degradation upon fusion with lysosomes, which makes it quite challenging for detection. There are several methods for detection in mammalian cells, including immunoblotting analysis of LC3 and p62 and detection of autophagosome formation/maturation by fluorescence microscopy, Currently, there is no single “gold standard” for determining the autophagic activity that is applicable in every experimental context, hence it is recommended to go for the combined use of multiple methods to accurately assess the autophagic activity in any given biological setting.

Get tips on using ON-TARGETplus Human NOS2 (4843) siRNA - SMARTpool to perform siRNA / miRNA gene silencing Human - Caco-2 iNOS/NOS2

Get tips on using ON-TARGETplus Human CFL1 (1072) siRNA - SMARTpool to perform siRNA / miRNA gene silencing Human - MDA-MB-231 CFL1

Get tips on using ON-TARGETplus Human NBN (4683) siRNA - SMARTpool to perform siRNA / miRNA gene silencing Human - MCF-7 NBS1/NBN

Get tips on using ON-TARGETplus Human RAD51 (5888) siRNA - SMARTpool to perform siRNA / miRNA gene silencing Human - MDA-MB-231 RAD51

Get tips on using ON-TARGETplus Human AHR (196) siRNA - Individual to perform siRNA / miRNA gene silencing Human - MDA-MB-231 AHR

Get tips on using ON-TARGETplus Human PCSK6 (5046) siRNA - Individual to perform siRNA / miRNA gene silencing Human - Detroit 562 / D562 PACE4

Get tips on using ON-TARGETplus Human FURIN (5045) siRNA - SMARTpool to perform siRNA / miRNA gene silencing Human - Detroit 562 / D562 Furin

Get tips on using ON-TARGETplus Human PCSK7 (9159) siRNA - Individual to perform siRNA / miRNA gene silencing Human - Detroit 562 / D562 PC7