ELISA (kit) IL-6, IL-1β, IL-8 and TNF-α -NA- Human

Get tips on using Brilliant Violet 605™ anti-human CD69 Antibody to perform Flow cytometry Anti-bodies Human - CD69

Get tips on using Total RNA Purification Kit to perform RNA isolation / purification Tissue - Human Adrenal glands

Get tips on using RNeasy Plus Mini Kit to perform RNA isolation / purification Tissue - Human Adrenal glands

ROS has a very short half-lives in biological environment as they are influenced by exposure to ambient oxygen. As it is highly reactive and hard to measure care should be taken to ensure the stability of the sample during isolation, preparation, storage, and analysis.

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 AquaRNA Kit to perform RNA isolation / purification Cells - primary human mesenchymal stem cells

Get tips on using Alexa Fluor® 647 Mouse Anti-Human CD24 to perform Flow cytometry Anti-bodies Human - CD24

Get tips on using Live and Dead Cell Assay (Abcam) to perform Live / Dead assay mammalian cells - MC3T3

Get tips on using Live and Dead Cell Assay (Abcam) to perform Live / Dead assay yeast - Syspastospora parasitica

Get tips on using Live and Dead Cell Assay (Abcam) to perform Live / Dead assay yeast - Saprolegnia diclina