ChIP H3K36Me3 Canine Mouse

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RNA isolation / purification Yeast - Coprinus cinereus Experiment

Generally it has been difficult to isolate high-quality RNA from yeast because of problems disrupting the cells. Use of enzymes to disrupt cell wall can alter gene expression profiles. Therefore, physical disruption can result in high quality RNA for all downstream processing. Use of DNAse and proteinase K will remove traces of DNA contamination and proteins respectively.

RNA RNA isolation / purification Yeast Coprinus cinereus

shRNA gene silencing Human - Neuroblastoma cells (SH-SY5Y) Connexin 43 lentiviral particles Experiment

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.

RNA shRNA gene silencing Human Neuroblastoma cells (SH-SY5Y) Connexin 43 lentiviral particles

TRI Reagent® MRC Product

Get tips on using TRI Reagent® MRC to perform RNA isolation / purification Yeast - Coprinus cinereus

Products Molecular Research Center, Inc. TRI Reagent® MRC

RNeasy Plant Mini Kit Product

Get tips on using RNeasy Plant Mini Kit to perform RNA isolation / purification Yeast - Coprinus cinereus

Products Qiagen RNeasy Plant Mini Kit

PolyATtract® mRNA Isolation Systems Product

Get tips on using PolyATtract® mRNA Isolation Systems to perform RNA isolation / purification Yeast - Coprinus cinereus

Products Promega PolyATtract® mRNA Isolation Systems

Muse® Cell Cycle Assay Kit Product

Get tips on using Muse® Cell Cycle Assay Kit to perform Cell cycle assay human - SW480