Housekeeping Gene Selection Advisory: Glyceraldehyde-3-Phosphate Dehydrogenase and [beta]-Actin Are Targets of miR-644a

Results of overexpression or downregulation of a microRNA (miRNA) on its target mRNA expression are often validated by reverse-transcription and quantitative PCR analysis using an appropriate housekeeping gene as an internal control. The possible direct or indirect effects of a miRNA on the expression of housekeeping genes are often overlooked. Among many housekeeping genes, expressions of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and [beta]-actin have been used extensively for normalization of gene expression data. Here, we show that GAPDH and [beta]-actin are direct targets of miR-644a. Our data demonstrate the unsuitability of GAPDH and [beta]-actin as internal controls in miR-644a functional studies and emphasize the need to carefully consider the choice of a reference gene in miRNA experiments.