DNA methylation profiling Gene specific profiling Mouse muscle stem cells

Get tips on using GeneJET RNA Purification Kit to perform RNA isolation / purification Cells - primary human renal artery smooth muscle 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 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 Neuron specific enolase monoclonal antibody (47) to perform Immunohistochemistry Mouse - NSE

Get tips on using Lipofectamine® 2000 Transfection Reagent to perform DNA transfection Mammalian cells - Primary cells Rat mesenchymal stem cells (rMSC)

Get tips on using pGL3-Basic Vector to perform Reporter gene assay luciferase - human embryonic stem cells

Get tips on using CellGenix® GMP SCGM Stem Cell Growth Medium to perform Mammalian cell culture media NK-92

Get tips on using TRIzol Reagent to perform RNA isolation / purification Cells - primary rabbit aortic smooth muscle cells

Get tips on using TRIzol Reagent to perform RNA isolation / purification Cells - primary human aortic smooth muscle cells

DNA microarrays enable researchers to monitor the expression of thousands of genes simultaneously. However, the sensitivity, accuracy, specificity, and reproducibility are major challenges for this technology. Cross-hybridization, combination with splice variants, is a prime source for the discrepancies in differential gene expression calls among various microarray platforms. Removing (either from production or downstream bioinformatic analysis) and/or redesigning the microarray probes prone to cross-hybridization is a reasonable strategy to increase the hybridization specificity and hence, the accuracy of the microarray measurements.

Get tips on using RNeasy Mini Kit to perform RNA isolation / purification Cells - primary rabbit skeletal muscle cells