Quantitative real-time reverse transcription polymerase chain reaction: normalization to rRNA or single housekeeping genes is inappropriate for human tissue biopsies

Carmela Tricarico; Pamela Pinzani; Simonetta Bianchi; Milena Paglierani; Vito Distante; Mario Pazzagli; Stephen A Bustin; Claudio Orlando

doi:10.1016/s0003-2697(02)00311-1

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.

Authors

Carmela Tricarico¹, Pamela Pinzani, Simonetta Bianchi, Milena Paglierani, Vito Distante, Mario Pazzagli, Stephen A Bustin, Claudio Orlando

Affiliation

¹ Clinical Biochemistry Unit, Department of Clinical Physiopathology, University of Florence, Florence, Italy.

PMID: 12413463
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