siRNA / miRNA gene silencing Human HNSCC cell line

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.

Get tips on using Glut1 siRNA and shRNA Plasmids (h) to perform RNA sequencing Human - HT-1376 (urinary bladder cell line)

Get tips on using CD74 siRNA and shRNA Plasmids (h) to perform RNA sequencing Human - HT-1376 (urinary bladder cell line)

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 In Situ Cell Death Detection Kit, Fluorescein to perform TUNEL assay cell type - HNSCC Detroit 562 human head and neck tumor cells

Get tips on using Rock-2 siRNA and shRNA Plasmids (h) to perform RNA sequencing Human - HT-1376 (urinary bladder cell line)

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

Hello Iam a phd student in pharmacy and i want to know if this technology is suitable to knockout or silencing part of the gas5 gene in BV2 cells please

Get tips on using Slc1a2 to perform siRNA / miRNA gene silencing Rat - Glial cells GLT-1

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.