Evaluation of reference genes for quantitative PCR analysis of mouse lymphocytes

J Immunol Methods. 2012 Oct 31;384(1-2):196-9. doi: 10.1016/j.jim.2012.07.020. Epub 2012 Aug 4.

Abstract

Normalization to a reference gene is the method of choice for quantitative PCR analysis. The stability of reference genes is critical for accurate gene expression analysis, as significant variations in reference gene expression can alter experimental results and conclusions. In this study, we evaluated the expression stability of five commonly used reference genes found in mouse lymphocytes. Using NormFinder and BestKeeper algorithms, we consistently show that ubiquitin C (Ubc) is the optimal reference gene for normalizing qPCR data obtained from mouse lymphocytes, whereas beta-actin (Actb) is not a suitable reference gene due to its extensive variability in expression. Our findings emphasize the importance of validating reference genes for qPCR analyses. We provide a shortlist of reference genes to use for normalization and recommend freely available software programs as a rapid approach to validate potential reference genes.

Publication types

  • Evaluation Study
  • Research Support, N.I.H., Extramural

MeSH terms

  • 14-3-3 Proteins / genetics
  • Actins / genetics
  • Animals
  • B-Lymphocytes / metabolism
  • Gene Expression Profiling / methods
  • Gene Expression Profiling / standards*
  • Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) / genetics
  • Hypoxanthine Phosphoribosyltransferase / genetics
  • Lymphocytes / metabolism*
  • Mice
  • Mice, Inbred C57BL
  • Reference Standards
  • Reproducibility of Results
  • Reverse Transcriptase Polymerase Chain Reaction / methods
  • Reverse Transcriptase Polymerase Chain Reaction / standards*
  • Software*
  • Ubiquitin C / genetics

Substances

  • 14-3-3 Proteins
  • Actins
  • Ubiquitin C
  • YWHAZ protein, human
  • Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)
  • Hypoxanthine Phosphoribosyltransferase