siRNA / miRNA gene silencing Human Primary Endometrial Stromal Cells hsa-miR-542-3p

Get tips on using AMPKα1/2 siRNA (h) to perform siRNA / miRNA gene silencing Human - HepG2 AMPKα1/α2

Get tips on using CREB-2 siRNA (h) to perform siRNA / miRNA gene silencing Human - HUVEC ATF4 Lipid

Get tips on using SignalSilence® SAPK/JNK siRNA to perform siRNA / miRNA gene silencing Human - KGN SAPK/JNK

Get tips on using SMARTpool: ON-TARGETplus TP63 siRNA to perform siRNA / miRNA gene silencing Human - A253 P36

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 B2M siRNA to perform siRNA / miRNA gene silencing Human - hES cell line H1 (WA01) B2M

Get tips on using Ccl2 siRNA to perform siRNA / miRNA gene silencing Rat - Neuronal cells MCP-1

Get tips on using Cxcr4 siRNA to perform siRNA / miRNA gene silencing Mouse - Embryonic stem cells CXCR4

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 TRIzol Reagent to perform RNA isolation / purification Cells - primary human marrow stromal cells