Microarray Gene expression arrays Human whole blood cells

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 have 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 efficacy of transduction and shRNA on its target site.

Get tips on using DNeasy Blood & Tissue Kit to perform DNA isolation / purification Bacteria - Gram positive piezophilic bacteria [AT7 and AT12 Strains]

Get tips on using BLOOD AGAR BASE NO.2 to perform Bacterial cell culture media Helicobacter pylori

Get tips on using EZ1 DNA Blood 200 µl Kit (48) to perform DNA isolation / purification Tissue - blood / plasma

Get tips on using QIAamp DNA Blood BioRobot MDx Kit (12) to perform DNA isolation / purification Tissue - blood / plasma

Get tips on using QIAamp DNA Blood BioRobot 9604 Kit (12) to perform DNA isolation / purification Tissue - blood / plasma

Get tips on using QIAamp DNA Blood Mini Kit to perform DNA isolation / purification Tissue - eye

Get tips on using QIAamp DNA Blood Mini Kit to perform DNA isolation / purification Yeast - Saccharomyces cerevisiae

Get tips on using QIAamp DNA Blood Mini Kit to perform DNA isolation / purification Bacteria - Gram positive Staphylococcus aureus

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.