Housekeeping gene selection advisory: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin are targets of miR-644a

PLoS One. 2012;7(10):e47510. doi: 10.1371/journal.pone.0047510. Epub 2012 Oct 16.

Abstract

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 β-actin have been used extensively for normalization of gene expression data. Here, we show that GAPDH and β-actin are direct targets of miR-644a. Our data demonstrate the unsuitability of GAPDH and β-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.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • 3' Untranslated Regions
  • Actins / genetics*
  • Base Pairing
  • Base Sequence
  • Cell Line
  • Gene Expression Regulation
  • Gene Order
  • Genes, Essential
  • Glyceraldehyde-3-Phosphate Dehydrogenases / genetics*
  • Humans
  • MicroRNAs / genetics*
  • MicroRNAs / metabolism
  • Molecular Sequence Data
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • Sequence Alignment

Substances

  • 3' Untranslated Regions
  • Actins
  • MIRN644 microRNA, human
  • MicroRNAs
  • RNA, Messenger
  • Glyceraldehyde-3-Phosphate Dehydrogenases

Associated data

  • RefSeq/NM_001101
  • RefSeq/NM_002046

Grants and funding

Research in GCS laboratory is supported by grants from National Science Foundation (NSF) and DoD Prostate Cancer Research Program. JS was supported by NSF grant 0842606 and JSE was supported by DoD-USAMRAA grant W81XWH-11-1-0204. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.