DNA Damage Assay Human bronchial epithelial cells (hBE)

Get tips on using Human Candida Albicans ELISA Kit to perform Cell Culture Contamination Detection Kit Yeast

Get tips on using MammoCult™ Human Medium Kit to perform 3D Cell Culture Media PDX mammospheres

Get tips on using Cell Counting Kit-8 to perform Live / Dead assay mammalian cells - GH3

Gene silencing through the use of small interfering RNA (siRNA) has become a primary tool for identifying disease-causing genes. There are several aspects for preparing and delivering effective siRNA to knockdown a target gene. The length of siRNA should be 21–23nt long with G/C content 30–50%. If a validated siRNA sequence for your target gene is not available, use siRNA generated against the entire target gene ORF. Always work with two or three different siRNA constructs to get reliable results. If you are not sure how much siRNA to use for a given experiment, start with a transfection concentration of 10-50 nM and use siRNA-specific transfection reagent to ensure efficient siRNA delivery in a wide range of cells.

Get tips on using Dynabeads™ Untouched™ Human Monocytes Kit to perform Cell Isolation Monocyte

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 Accell Human VDAC1 (7416) siRNA - Set of 4 to perform siRNA / miRNA gene silencing Human - HeLa VDAC1

Get tips on using Accell Human VDAC1 (7416) siRNA - Set of 4 to perform siRNA / miRNA gene silencing Human - HEK293 VDAC1

Get tips on using Accell Human VDAC1 (7416) siRNA - Set of 4 to perform siRNA / miRNA gene silencing Human - HepG2 VADC1