mRNA / Ribonucleoprotein isolation / purification

Get tips on using ON-TARGETplus Human RAB11FIP1 (80223) siRNA - SMARTpool to perform siRNA / miRNA gene silencing Human - MDA-MB-231 Rab Coupling Protein (RCP)

Get tips on using Silencer® Select Negative Control No 1 siRNA to perform siRNA / miRNA gene silencing Mouse - siRNA negative control polymer / lipid

Get tips on using Lipofectamine® 2000 Transfection Reagent to perform siRNA / miRNA gene silencing Human - Primary Endometrial Stromal Cells IGFBP1 (Insuline-like growth factor binding protein-1) Lipid

Get tips on using heat shock protein family A (Hsp70) member 5 to perform siRNA / miRNA gene silencing Human - PC3 (human prostate cancer cell line) HSPA5 (GRP78)

Get tips on using SiRNA silencing human Eph receptor B4, Id: s243 to perform siRNA / miRNA gene silencing Human - HNSCC cell line Eph receptor B4 Polymer / Lipid

Get tips on using SiRNA silencing human Eph receptor B4, Id: 533 to perform siRNA / miRNA gene silencing Human - HNSCC cell line Eph receptor B4 Polymer / Lipid

Get tips on using GenomONE™-Neo HVJ-E Membrane Fusion Transfection Kit to perform siRNA / miRNA gene silencing Human - U937 MK2 (MAPK Kinase 2) Viral vectors

Get tips on using GenomONE™-Neo HVJ-E Membrane Fusion Transfection Kit to perform siRNA / miRNA gene silencing Human - Jurkat MK2 (MAPK Kinase 2) Viral vectors

Get tips on using Pre-designed and validated siRNA against gene IGFBP1 to perform siRNA / miRNA gene silencing Human - Primary Endometrial Stromal Cells IGFBP1 (Insuline-like growth factor binding protein-1)

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.