siRNA / miRNA gene silencing Rat MTLn3 (rat mammary adenocarcinoma breast cancer cell line)

Get tips on using DMEM/F-12, no phenol red to perform 3D Cell Culture Media Human breast cancer MDA-MB-231 cells-Mammospheres

Get tips on using DMEM/F-12 PLUS Basal Medium to perform 3D Cell Culture Media Human breast cancer MDA-MB-231 cells-Mammospheres

Get tips on using Fenozol to perform siRNA / miRNA gene silencing Human - HeLa Cdc20

Get tips on using SIHK1738 to perform siRNA / miRNA gene silencing Human - LLC PKN3

Get tips on using SIHK1738 to perform siRNA / miRNA gene silencing Human - HeLa PKN3

Get tips on using NM_001350 to perform siRNA / miRNA gene silencing Human - U2OS DAXX

Get tips on using FGD4 siRNAs (FlexiTube) to perform siRNA / miRNA gene silencing Human - LNCap FGD4

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 TurboFect Transfection Reagents to perform siRNA / RNAi /miRNA transfection Rat - A-10 Cationic lipid based

Get tips on using HiPerFect Transfection Reagent to perform siRNA / RNAi /miRNA transfection Rat - A-10 Cationic lipid based