siRNA / miRNA gene silencing Human PC3 (human prostate cancer cell line)

Get tips on using Accell Human VEGFC (7424) siRNA - SMARTpool to perform siRNA / miRNA gene silencing Human - LAMA84 VEGFC

Get tips on using Accell Human MYB (4602) siRNA - SMARTpool to perform siRNA / miRNA gene silencing Human - K562 MYB

Get tips on using Accell Human VEGFC (7424) siRNA - SMARTpool to perform siRNA / miRNA gene silencing Human - K562 VEGFC

Get tips on using Accell Human CD44 (960) siRNA - SMARTpool to perform siRNA / miRNA gene silencing Human - HaCaT CD44

Get tips on using ON-TARGETplus Human PCSK7 (9159) siRNA - Individual to perform siRNA / miRNA gene silencing Human - Detroit 562 / D562 PC7

Get tips on using ON-TARGETplus Human PCSK7 (9159) siRNA - Individual to perform siRNA / miRNA gene silencing Human - A253 PC-7

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 ON-TARGETplus Human PCSK7 (9159) siRNA - Individual to perform siRNA / miRNA gene silencing Human - A253 IGFBP-7

Get tips on using SNP Type™ 96.96 Genotyping Reagent Kit with Control Line Fluid—10 IFCs to perform Cell line authentication Human prostatic cancer cell line DU145

The RNA interference (RNAi) is used to inhibit gene expression or translation, by neutralizing targeted mRNA molecules. Two types of RNA molecules such as microRNA (miRNA) and small interfering RNA (siRNA) play a central role in RNAi. Few points have to considered to increase the transfection efficiency of siRNA. Always use healthy, actively dividing cells to maximize transfection efficiency. The confluency of cells should be between 50-70%. Always use the most appropriate siRNA concentration to avoid off-target effects and unwanted toxic side effects. Positive and negative controls should be used for each and every experiment to determine transfection efficiency.