DNA transfection Mammalian cells Primary cells

Get tips on using RIPA Lysis Buffer System to perform Protein isolation Mammalian cells - HOG

Get tips on using RIPA Lysis Buffer, 10X to perform Protein isolation Mammalian cells - STTG1

Get tips on using 2-D Quant Kit to perform Protein quantification Mammalian cells - SiHa

Get tips on using Qproteome Mammalian Protein Prep Kit to perform Protein isolation Tissue - Human tissue C-MFPE samples

Get tips on using Mammalian beta-Galactosidase Assay Kit to perform Reporter gene assay β-galactosidase substrates - Aspc-1

Get tips on using Mammalian beta-Galactosidase Assay Kit to perform Reporter gene assay β-galactosidase substrates - Bxpc-3

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 DCFDA - Cellular Reactive Oxygen Species Detection Assay Kit to perform ROS assay cell type - human primary corneal epithelial cells

Get tips on using TaqMan® MicroRNA Reverse Transcription Kit to perform siRNA / RNAi /miRNA transfection Mouse - Glomerular Mesangial cells polymer / lipid

Get tips on using Rock-2 siRNA and shRNA Plasmids (h) to perform siRNA / RNAi /miRNA transfection Human Cells - HT-1376 ROCK2