Quantitative Real-Time Reverse Transcription Polymerase Chain Reaction: Normalization to rRNA or Single Housekeeping Genes Is Inappropriate for Human Tissue Biopsies

Anal Biochem. 2002 Oct 15;309(2):293-300. doi: 10.1016/s0003-2697(02)00311-1.

Abstract

Careful normalization is essential when using quantitative reverse transcription polymerase chain reaction assays to compare mRNA levels between biopsies from different individuals or cells undergoing different treatment. Generally this involves the use of internal controls, such as mRNA specified by a housekeeping gene, ribosomal RNA (rRNA), or accurately quantitated total RNA. The aim of this study was to compare these methods and determine which one can provide the most accurate and biologically relevant quantitative results. Our results show significant variation in the expression levels of 10 commonly used housekeeping genes and 18S rRNA, both between individuals and between biopsies taken from the same patient. Furthermore, in 23 breast cancers samples mRNA and protein levels of a regulated gene, vascular endothelial growth factor (VEGF), correlated only when normalized to total RNA, as did microvessel density. Finally, mRNA levels of VEGF and the most popular housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were significantly correlated in the colon. Our results suggest that the use of internal standards comprising single housekeeping genes or rRNA is inappropriate for studies involving tissue biopsies.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biopsy
  • Breast Neoplasms / blood supply
  • Breast Neoplasms / genetics*
  • Breast Neoplasms / metabolism*
  • Breast Neoplasms / surgery
  • Colonic Neoplasms / blood supply
  • Colonic Neoplasms / genetics*
  • Colonic Neoplasms / metabolism*
  • Colonic Neoplasms / surgery
  • Endothelial Growth Factors / chemistry
  • Endothelial Growth Factors / genetics*
  • Endothelial Growth Factors / metabolism
  • Female
  • Gene Expression Regulation
  • Glyceraldehyde-3-Phosphate Dehydrogenases / genetics
  • Glyceraldehyde-3-Phosphate Dehydrogenases / metabolism
  • Humans
  • Immunohistochemistry
  • Intercellular Signaling Peptides and Proteins / chemistry
  • Intercellular Signaling Peptides and Proteins / genetics*
  • Intercellular Signaling Peptides and Proteins / metabolism
  • Lymphokines / chemistry
  • Lymphokines / genetics*
  • Lymphokines / metabolism
  • Male
  • Neovascularization, Pathologic / genetics
  • RNA, Messenger / analysis*
  • RNA, Messenger / biosynthesis
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • RNA, Neoplasm / analysis
  • RNA, Neoplasm / genetics
  • RNA, Neoplasm / metabolism
  • RNA, Ribosomal, 18S / analysis
  • RNA, Ribosomal, 18S / genetics
  • RNA, Ribosomal, 18S / metabolism
  • Reference Standards
  • Reproducibility of Results
  • Reverse Transcriptase Polymerase Chain Reaction*
  • Vascular Endothelial Growth Factor A
  • Vascular Endothelial Growth Factors

Substances

  • Endothelial Growth Factors
  • Intercellular Signaling Peptides and Proteins
  • Lymphokines
  • RNA, Messenger
  • RNA, Neoplasm
  • RNA, Ribosomal, 18S
  • Vascular Endothelial Growth Factor A
  • Vascular Endothelial Growth Factors
  • Glyceraldehyde-3-Phosphate Dehydrogenases