siRNA / miRNA gene silencing Rat IEC-6

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.

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 PhosphoProtein Purification Kit (6) to perform Protein tag Purification of phosphorylated proteins

Get tips on using STEAP2 metalloreductase to perform siRNA / miRNA gene silencing Human - PC3 (human prostate cancer cell line) STEAP2

Get tips on using X-tremeGENE™ siRNA Transfection Reagent to perform siRNA / RNAi /miRNA transfection Rat - AR42J Lipid based

Get tips on using 3D-Gene® Mouse miRNA Oligo chip (ver.21) to perform Microarray Gene expression arrays - Mouse liver tissue Cyanine-3-CTP

Get tips on using pcDNA™3.1 (+) Mammalian Expression Vector to perform siRNA / miRNA gene silencing Human - U251 cofilin-1 (CFL1)

Get tips on using MISSION® esiRNA_esiRNA targeting mouse Lrp5 (esiRNA1) to perform siRNA / miRNA gene silencing Mouse - MLO‐Y4 Lrp5

Get tips on using MISSION® esiRNA_esiRNA targeting mouse Lrp6 (esiRNA1) to perform siRNA / miRNA gene silencing Mouse - MLO‐Y4 Lrp6

Get tips on using anti-Mucin 6, Clone: CLH5, Invitrogen™ to perform Immunohistochemistry Human - MUC-6