Comparing and measuring gene expression is certainly an integral part of research—gene expression patterns continue to show us how different cell networks are regulated, and point to new biological pathways and possible treatments for disease.
But one crucial part of gene expression lies in making sure that differences in gene expression are due to gene expression, and NOT variations in the amount of starting material, enzyme efficiencies, and overall transcription levels in different cells and tissues. What can control for all that? Reference genes.
Reference genes, or “housekeeping” genes, are those that express at a constant, known level, regardless of what do you in your experiments. By comparing your test samples reference genes, you can compare RT-PCR results between different types of cells or tissues, and not have to worry about differing sample sizes.
So how do you pick out a reference gene?
One nice thing about reference genes is that you can normalize results with very little starting material (often an issue with RT-PCR analysis). By comparing Ct values of your target and reference genes, you can set normal values of gene expression without calculating mass or other values.
Anybody have a favorite set of reference genes?
Bio-Rad (2012). Relative quantification normalized against a reference gene. Gene Expression Gateway.
Vandesompele, J., et al. (2002). Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biology, 3(7), research0034.1-0034.11.
Zhang, X., et al. (2005). Selection of reference genes for gene expression studies in human neutrophils by real-time PCR. BMC Molecular Biology, 6, 4.Photo Credits