siRNA / miRNA gene silencing Human PANC-1

Get tips on using GenomONE™-Neo HVJ-E Membrane Fusion Transfection Kit to perform siRNA / miRNA gene silencing Human - Jurkat MK2 (MAPK Kinase 2) Viral vectors

Hello Iam a phd student in pharmacy and i want to know if this technology is suitable to knockout or silencing part of the gas5 gene in BV2 cells please

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 MicroRNA isolation kit to perform siRNA / miRNA gene silencing Rat - IEC-6 HuR

Get tips on using β-Gal Assay Kit to perform Reporter gene assay β-galactosidase substrates - PANC-1

Get tips on using Human TIM-1/KIM-1/HAVCR DuoSet ELISA to perform ELISA Human - KIM-1

Get tips on using Purified Mouse Anti-Human ZO-1 Clone 1/ZO-1 (RUO) to perform Western blotting ZO-1

Reporter gene assays enable high sensitivity measurement of gene expression and cell signaling through the addition of bioluminescent genes into target cells. One of the major challenges is to make a specific construct that has no responses other than those related to the signaling pathway of interest. This can be achieved by selecting highly specific reporter constructs containing only defined responsive elements and a minimal promoter linked to reporter enzymes such as luciferase

Get tips on using ChIP Kit Magnetic - One Step (ab156907) to perform ChIP Human - PANC-1

Get tips on using Beta-Glo® Assay System to perform Reporter gene assay β-galactosidase substrates - PANC-1