siRNA / miRNA gene silencing Human HT-29

Get tips on using Slc1a2 to perform siRNA / miRNA gene silencing Rat - Glial cells GLT-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 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.

Get tips on using Glut1 siRNA and shRNA Plasmids (h) to perform RNA sequencing Human - HT-1376 (urinary bladder cell line)

Get tips on using CD74 siRNA and shRNA Plasmids (h) to perform RNA sequencing Human - HT-1376 (urinary bladder cell line)

Get tips on using MicroRNA isolation kit to perform siRNA / miRNA gene silencing Rat - IEC-6 HuR

Get tips on using QCM ECMatrix Cell Invasion Assay, 24-well (8 µm), fluorimetric to perform Cell migration / Invasion cell type - HT-29

Get tips on using Rock-2 siRNA and shRNA Plasmids (h) to perform RNA sequencing Human - HT-1376 (urinary bladder cell line)

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

Cell Invasion or Cell Migration assays are technically challenging to set up as the gradient between the two compartments equilibrates in time during the assay. It is also problematic to view cells and for cells to migrate through a non-physiologic polycarbonate or polypropylene filter. Care must be taken while loading the well with cells to form a single cell suspension. Precaution must be taken while trypsinization (under-trypsinization can lead to cell clumping while over-trypsinization could strip off adhesion molecules necessary for migration). This leads to difficulty in getting significant results, when only small numbers of cells cross the filter or when the distribution and/or staining of the cells is uneven.

Get tips on using FlexiTube GeneSolution GS27279 for Tnfrsf12a to perform siRNA / miRNA gene silencing Mouse - B16-BL6 FN14/Tnfrsf12a