DNA transfection Mammalian cells Immortalized cell lines

Get tips on using Senescence Cells Histochemical Staining Kit to perform Reporter gene assay β-galactosidase substrates - CHO

Get tips on using Senescence Cells Histochemical Staining Kit to perform Reporter gene assay β-galactosidase substrates - Hep3B

Get tips on using Senescence Cells Histochemical Staining Kit to perform Reporter gene assay β-galactosidase substrates - HepG2

Get tips on using Senescence Cells Histochemical Staining Kit to perform Reporter gene assay β-galactosidase substrates - A549

Get tips on using MagCellect Human B Cell Isolation Kit to perform Cell Isolation B cell

Get tips on using B Cell Isolation Kit II, human to perform Cell Isolation B cell

Get tips on using Cell-APOPercentage™ Apoptosis Assay to perform Apoptosis assay cell type - SKOV3

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.

Generally it has been difficult to isolate high-quality RNA from yeast because of problems disrupting the cells. Use of enzymes to disrupt cell wall can alter gene expression profiles. Therefore, physical disruption can result in high quality RNA for all downstream processing. Use of DNAse and proteinase K will remove traces of DNA contamination and proteins respectively.