rna-isolation-purification-cells-primary-mouse-dorsal-root-ganglion-neurons

Get tips on using HiPurA™ Streptomyces DNA Purification Kit to perform DNA isolation / purification Bacteria - Gram positive Streptomyces. Sp

Get tips on using Wizard® Genomic DNA Purification Kit to perform DNA isolation / purification Bacteria - Gram positive Enterococcus faecium

Get tips on using Wizard® Genomic DNA Purification Kit to perform DNA isolation / purification Bacteria - Gram positive Bacillus subtilis

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.

Transfection is a powerful technique that enables the study of the function of genes and gene products in cells. Based on the nature of experiments, we may need a stable DNA transfection in cells for persistent gain-of-function or loss-of-function of the target gene. For stable transfection, integration of a DNA vector into the chromosome is crucial which requires selective screening and clonal isolation. By carefully selecting a viral delivery system and related reagents we can ensure safe and highly-efficient delivery of expression constructs for high-level constitutive or inducible expression in any mammalian cell type.

Get tips on using Lipofectamine® 2000 Transfection Reagent to perform DNA transfection Mammalian cells - Primary cells Rat dermal fibroblasts (rDF)

Get tips on using PowerSoil® DNA isolation to perform DNA isolation / purification Bacteria - Gram positive Enterococcus faecium

Get tips on using PowerSoil® DNA isolation to perform DNA isolation / purification Bacteria - Gram positive Bacillus subtilis

Get tips on using TruSeq Stranded Total RNA to perform RNA sequencing Mouse - RAW264.7

Get tips on using QIAamp MinElute Virus Vacuum Kit (50) to perform RNA isolation / purification Viral - Viral CNS disease