RNA quantification

Get tips on using miRNeasy Serum/Plasma Advanced Kit (50) to perform RNA isolation / purification Tissue - Livestock Blood / Serum / Plasma / Buffy coat

Get tips on using Quick Amp Labeling Kit-one color to perform Microarray RNA amplification & Labeling - Mouse Myofibers Cy3- or/and Cy5

Get tips on using Rock-2 siRNA and shRNA Plasmids (h) to perform RNA sequencing Human - HT-1376 (urinary bladder cell line)

Get tips on using mirVana™ miRNA Isolation Kit, with phenol to perform RNA isolation / purification Cells - primary human airway epithelial cells

Get tips on using mirVana™ miRNA Isolation Kit, with phenol to perform RNA isolation / purification Cells - primary human aortic endothelial cells

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 GeneChip® HT 3' IVT PLUS Reagent Kit to perform Microarray Human - Precision cut lung slices Target preparation kit (RNA Amplification + Hybridization + control)

Get tips on using Low Input Quick Amp Labeling Kits to perform Microarray RNA amplification & Labeling - Human endometrial stromal cells Cyanine 3-pCp

Get tips on using Low Input Quick Amp Labeling Kits to perform Microarray RNA amplification & Labeling - Fish fundulus heteroclitus Cyanine-3 / Cyanine-5

Get tips on using miRNA Complete Labeling and Hyb Kit to perform Microarray RNA amplification & Labeling - Human endometrial stromal cells Cyanine 3-pCp