Microarray Mice Cochlea

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 RNeasy MinElute Cleanup Kit to perform RNA isolation / purification Tissue - Human Kidney

Get tips on using QIAamp MinElute Media Kit to perform DNA isolation / purification Tissue - genital / cervical samples

Get tips on using QIAamp MinElute Virus Spin Kit (50) to perform DNA isolation / purification Viral

Get tips on using MinElute PCR Purification Kit to perform DNA gel extraction / PCR product purification Product size < 15Kb

Get tips on using QIAamp MinElute ccfDNA Mini Kit (50) to perform DNA isolation / purification Tissue - blood / plasma

Get tips on using MinElute Reaction Cleanup Kit (250) to perform DNA gel extraction / PCR product purification Product size < 15Kb

Get tips on using MinElute Gel Extraction Kit (250) to perform DNA gel extraction / PCR product purification Product size < 15Kb

Get tips on using MinElute 96 UF PCR Purification Kit (24) to perform DNA isolation / purification Plasmid purification