siRNA / miRNA gene silencing Human U937 MK2 (MAPK Kinase 2)

Get tips on using Glut1 siRNA and shRNA Plasmids (h) to perform siRNA / miRNA gene silencing Human - HT-1376 GLUT1

Get tips on using CD74 siRNA and shRNA Plasmids (h) to perform siRNA / miRNA gene silencing Human - HT-1376 CD74

Get tips on using siRNA ATX-1 or ENPP2 to perform siRNA / miRNA gene silencing Human - A2780 ATX-1

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)

I would like to regulate the expression of a gene and in order to do that, I have purchased specific siRNA. After optimizing my transfection protocol and using electroporation I have achieved a 60-70% reduction of the gene of interest. However, I cannot observe a significant reduction of mRNA expression but only a reduction of protein. What might be the problem? Could the problem be in my cell treatment method?

Get tips on using Phospho-PI3 Kinase p85 (Tyr458)/p55 (Tyr199) Antibody to perform Autophagy assay cell type - HepG2

Get tips on using Anti-PI 3 Kinase p85 alpha antibody [EP380Y] to perform Autophagy assay cell type - HepG2

Get tips on using SIRT1 siRNA and shRNA Plasmids (h) to perform siRNA / miRNA gene silencing Human - PC3 (human prostate cancer cell line) SIRT1

Get tips on using Silencer select_ObR siRNA to perform siRNA / miRNA gene silencing Human - MCF-7 ObR(leptin receptor/LEPR)

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.