siRNA / miRNA gene silencing Human HeLa EPAS-1

RNAi or RNA interference is a common method to suppress gene expression in vitro/in vivo by utilizing the inherent microRNA machinery, without introducing a total gene knockout. miRNA is the inherent gene silencing machinery which can have more than one mRNA target, whereas siRNA can be designed to target a particular mRNA target. By design, both siRNA and miRNA are 20-25 nucleotides in length. The target sequence for siRNAs is usually located within the open reading frame, between 50 and 100 nucleotides downstream of the start codon. There are two ways in which cells can be transfected with desired RNAi: 1. Direct transfection (with calcium phosphate co-precipitation or cationic lipid-mediated transfection using lipofectamine or oligofectamine), and 2. Making RNAi lentiviral constructs (followed by transformation and transduction). Lentiviral constructs are time-consuming, but provide a more permanent expression of RNAi in the cells and consistent gene silencing. Direct transfection of oligonucleotides provides temporary genetic suppression. Traditional methods like calcium phosphate co-precipitation have challenges like low efficiency, poor reproducibility and cell toxicity. Whereas, cationic lipid-based transfection reagents are able to overcome these challenges, along with applicability to a large variety of eukaryotic cell lines.

Get tips on using ON-TARGETplus human ATG16L1 siRNA to perform siRNA / miRNA gene silencing Human - SHSY5Y ATG16L1

Get tips on using ON-TARGETplus Human TET1 siRNA to perform siRNA / miRNA gene silencing Human - A172 TET1

Get tips on using siGENOME Human DAB2 (1601) siRNA - SMARTpool to perform siRNA / miRNA gene silencing Human - A549 DAB2

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 CYP3A4 (1576) siRNA - SMARTpool to perform siRNA / miRNA gene silencing Human - Primary Human Hepatocytes CYP3A4

Get tips on using ON-TARGETplus Human CYP2B6 (1555) siRNA - SMARTpool to perform siRNA / miRNA gene silencing Human - Primary Human Hepatocytes CYP2B6

Get tips on using Silencer® Select Negative Control No 1 siRNA to perform siRNA / miRNA gene silencing Mouse - siRNA negative control polymer / lipid

Get tips on using SIHK1738 to perform siRNA / miRNA gene silencing Human - PANC-1 PKN3

Get tips on using s15827 to perform siRNA / miRNA gene silencing Human - PANC-1 CDC7