dna-methylation-profiling-gene-specific-profiling-ca-ski-hpv-16

Get tips on using PowerSoil® DNA isolation - DNeasy PowerSoil Pro Kit to perform DNA isolation / purification Bacteria - Gram positive Mycobacterium tuberculosis

Get tips on using PowerSoil® DNA isolation - DNeasy PowerSoil Pro Kit to perform DNA isolation / purification Bacteria - Gram positive Lactobacillus amylovorus

Get tips on using Quick-DNA™ Fungal/Bacterial Miniprep Kit to perform DNA isolation / purification Yeast - Candida albicans

Get tips on using T4 DNA Ligase (5 U/µL) to perform DNA ligation

Get tips on using Zymoclean™ Gel DNA Recovery Kit to perform DNA gel extraction / PCR product purification Product size > 15Kb

Get tips on using PowerSoil® DNA isolation to perform AAA for reviews

Get tips on using Quantifiler™ Duo DNA Quantification Kit to perform DNA quantification Blood

Get tips on using Quantifiler 1 Trio DNA Quantification Kit to perform DNA quantification Blood

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 Pre-designed and validated siRNA against gene IGFBP1 to perform siRNA / miRNA gene silencing Human - Primary Endometrial Stromal Cells IGFBP1 (Insuline-like growth factor binding protein-1)