Target gene expression levels are often normalized to the expression of a “housekeeping gene” (reference gene) to correct for differences in the amount of input RNA and variations in reaction efficiency. How do I select a suitable housekeeping gene?
Target gene expression levels are often normalized to the expression of a “housekeeping gene” (reference gene) to correct for differences in the amount of input RNA and variations in reaction efficiency. How do I select a suitable housekeeping gene?
There is no single, universally appropriate housekeeping gene suitable for accurate normalization in every experimental condition, as it is important to select housekeeping genes that do not vary in expression levels in the experimental system used. Common housekeeping genes that have been used in the past include GAPDH and ?-actin; however, reports in recent years indicate that expression of these genes may also vary, depending on the sample type and/or experimental conditions.
Using multiple housekeeping genes for normalization is currently the most reliable approach. In this strategy, the expression of multiple housekeeping genes is assayed, and the genes that show the lowest level of variation are selected for use. Software has been developed for selecting the optimum genes for correction (e.g., geNorm and BestKeeper). Inferences may also be made from microarray or RNA-seq expression profiling data, if available.
For additional information refer to: J. Vandesompele, et al. (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 3(7):RESEARCH0034.1–11.