Cell Isolation

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.

Reporter gene assays are designed to test the regulation of the expression of a gene of interest. This is usually done by linking the promoter of the gene of interest with a gene such as a firefly luciferase, which can be easily detected by addition of luciferin that leads to an enzymatic reaction to produce luminescence. The enzymatic reaction can be correlated to the expression of the gene of interest. Another luciferase gene that can be used is Renilla luciferase. For an appropriate luciferase assay: 1. the reporter should express uniformly in all cells, 2. specifically respond to effectors that the assay intends to monitor, 3. have low intrinsic stability to quickly reflect transcriptional dynamics. It is important to have an equal number of cells plated in each testing condition to avoid any incorrect readouts. Reporter assays could be single or dual reporter assays. The reporter could be both luciferases. Most dual-luciferase assays involve adding two reagents to each sample and measuring luminescence following each addition. Adding the first reagent activates the first luciferase reporter reaction; adding the second reagent extinguishes first luciferase reporter activity and initiates the second luciferase reaction. Dual-luciferase assays have some advantages, including 1. reduces variability, 2. reduces background, 3. normalizes differences in transfection efficiencies between samples.

Get tips on using Cellstain-Double Staining Kit to perform Live / Dead assay mammalian cells - A549

Get tips on using Cellstain-Double Staining Kit to perform Live / Dead assay mammalian cells - K562

Get tips on using Cellstain-Double Staining Kit to perform Live / Dead assay mammalian cells - TIG-118

Get tips on using Viromer® RED to perform DNA transfection Mammalian cells - Primary cells Rat schwann cells

Get tips on using Cellstain-Double Staining Kit to perform Live / Dead assay mammalian cells - HepaRG human hepatoma

Get tips on using SuperFect Transfection Reagent to perform DNA transfection Mammalian cells - Primary cells Human aortic smooth muscle cells (HOSMC)

Get tips on using FreeStyle™ 293-F Cells to perform Protein expression and purification Mammalian cells - HEK 293 EGFR

Get tips on using Lipofectamine® 2000 Transfection Reagent to perform DNA transfection Mammalian cells - Primary cells Rat mesangial cells