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

Get tips on using Accell Human VDAC1 (7416) siRNA - Set of 4 to perform siRNA / miRNA gene silencing Human - Min-6 VDAC1

Get tips on using Accell Human VDAC1 (7416) siRNA - Set of 4 to perform siRNA / miRNA gene silencing Human - PANC-1 VDAC1

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.

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.

RNA-Seq is a method to sequence RNA by applying Next Generation Sequencing (NGS). The quality of RNA is critical for the success of RNA-Seq. The integrity of RNA is measured by the RNA integrity number (RIN). RIN is computed from RNA electrophoresis and electropherogram profiles (the peak area of the 28S rRNA should be approximately twice the peak area of the 18S rRNA). If you get the RIN value lower than 7, the possibility of getting the low quality of RNA-seq data is high. To get a high quality RNA, it is better to work with fresh samples or snap-freeze the tissues in liquid nitrogen as quickly as possible and store them at -80°C until further use. Make sure designated areas and all your filter tips, microfuge tubes, plastic, and glassware are RNase-free.

Get tips on using ON-TARGETplus Human NCR3LG1 (374383) siRNA - SMARTpool to perform siRNA / miRNA gene silencing Human - HT-29 B7-H6/NCR3LG1

Get tips on using ON-TARGETplus Human ABL2 (27) siRNA - SMARTpool to perform siRNA / miRNA gene silencing Human - MDA-MB-231 ARG/ABL2

Get tips on using ON-TARGETplus Human SLC7A5 (8140) siRNA - Individual to perform siRNA / miRNA gene silencing Human - MDA-MB-231 LAT1/SLC7A5

Get tips on using ON-TARGETplus Human PLK1 (5347) siRNA - Individual to perform siRNA / miRNA gene silencing Human - MIA PaCa-2 PLK-1