Quantitative real-time reverse transcription polymerase chain reaction: normalization to rRNA or single housekeeping genes is inappropriate for human tissue biopsies.
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.
Tricarico
Carmela
C
Clinical Biochemistry Unit, Department of Clinical Physiopathology, University of Florence, Florence, Italy.
Pinzani
Pamela
P
Bianchi
Simonetta
S
Paglierani
Milena
M
Distante
Vito
V
Pazzagli
Mario
M
Bustin
Stephen A
SA
Orlando
Claudio
C
D001706:Biopsy; D001943:Breast Neoplasms / Q000098:blood supply / Q000235:genetics* / Q000378:metabolism* / Q000601:surgery; D003110:Colonic Neoplasms / Q000098:blood supply / Q000235:genetics* / Q000378:metabolism* / Q000601:surgery; D016228:Endothelial Growth Factors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005260:Female; D005786:Gene Expression Regulation; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D036341:Intercellular Signaling Peptides and Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008222:Lymphokines / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008297:Male; D009389:Neovascularization, Pathologic / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis* / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D012334:RNA, Neoplasm / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D012337:RNA, Ribosomal, 18S / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D012015:Reference Standards; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D016228:Endothelial Growth Factors; D036341:Intercellular Signaling Peptides and Proteins; D008222:Lymphokines; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D012337:RNA, Ribosomal, 18S; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases