siRNA / miRNA gene silencing Human HT-1080

Get tips on using HT 8-oxo-dG ELISA Kit II to perform DNA Damage Assay Capan-2

Get tips on using Lipofectamine® 2000 Transfection Reagent to perform siRNA / miRNA gene silencing Human - Primary Endometrial Stromal Cells hsa-miR-542-3p Lipid

Get tips on using HT 8-oxo-dG ELISA Kit II to perform DNA Damage Assay MIA PaCa-2

Get tips on using QIAamp 96 DNA QIAcube HT Kit (5) to perform DNA isolation / purification Tissue - blood / plasma

Get tips on using QIAamp 96 Virus QIAcube HT Kit (5) to perform RNA isolation / purification Viral - SARS-CoV-2

Get tips on using Fenozol to perform siRNA / miRNA gene silencing Rat - IEC-6 HuR

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 GenomONE™-Neo HVJ-E Membrane Fusion Transfection Kit to perform siRNA / miRNA gene silencing Human - U937 MK2 (MAPK Kinase 2) Viral vectors

Get tips on using GenomONE™-Neo HVJ-E Membrane Fusion Transfection Kit to perform siRNA / miRNA gene silencing Human - Jurkat MK2 (MAPK Kinase 2) Viral vectors

When extracting nucleic acids from cell cultures, thorough homogenization of cells via vortexing in lysis buffer is very necessary. Choose the best RNA isolation method keeping in mind the downstream applications, generally, column-based isolations result in clean and concentrated RNA samples. Downstream applications like sequencing and cDNA synthesis require high-quality RNA, always treat the samples with DNases and check their integrity by running a gel.