Systematic method for determining an ideal housekeeping gene for real-time PCR analysis.

To standardize the amount of biological material between samples (e.g., number of cells or amount of tissue) for quantitative real-time reverse transcriptase PCR (qRT-PCR), the cycle of the target gene at which expression is detected (the cycle threshold, or Ct) is divided by the Ct of a gene either thought to be unaffected by experimental conditions or similarly expressed among donors. Genes that maintain cellular structure or homeostasis, referred to as housekeeping genes, or 18S ribosomal RNA are often used for this purpose. Although unstable or inconsistent housekeeping gene expression will misrepresent experimental effects on target gene expression, housekeeping genes are often chosen arbitrarily rather than systematically. We designed a simple and systematic approach towards selection of housekeeping genes based on Ct variance (as reflected by the standard deviation) and normality of distribution. We validated this approach by comparing stability and consistency of expression of 11 housekeeping genes across different types of cells, experimental treatments, and human donors. Finally, we demonstrated the consequences of inconsistent housekeeping gene expression on the calculation of target gene expression, and conclude that validation of stability of housekeeping gene expression by considering both distribution normality and standard deviation is straightforward and critical for proper experimental design.