siRNA / miRNA gene silencing Human Melanoma cells (501 Mel and SK Mel 28)

Get tips on using ON-TARGET plus Mouse Becn1 (56208) siRNA - SMARTpool to perform siRNA / miRNA gene silencing Mouse - 4T1 BECN-1

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 SignalSilence® NF-κB p65 siRNA I #6261 to perform siRNA / miRNA gene silencing Rat - H9c2 NF-κB RelA (p65)

Get tips on using Lipofectamine® 2000 Transfection Reagent to perform siRNA / miRNA gene silencing Human - Primary Endometrial Stromal Cells IGFBP1 (Insuline-like growth factor binding protein-1) Lipid

Get tips on using pcDNA™3.1 (+) Mammalian Expression Vector to perform siRNA / miRNA gene silencing Human - U251 cofilin-1 (CFL1)

Get tips on using heat shock protein family A (Hsp70) member 5 to perform siRNA / miRNA gene silencing Human - PC3 (human prostate cancer cell line) HSPA5 (GRP78)

Get tips on using ON-TARGETplus Rat Snap23 (64630) siRNA - Set of 4 to perform siRNA / miRNA gene silencing Rat - RBL-2H3 Snap23

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 Phospho-SAPK/JNK (Thr183/Tyr185) (81E11) Rabbit mAb to perform siRNA / miRNA gene silencing Human - COV-434 SAPK/JNK

Get tips on using - to perform siRNA / miRNA gene silencing Rat - IEC-6