Validation of the comparative quantification method of real-time PCR analysis and a cautionary tale of housekeeping gene selection

McCurdy, Richard D., McGrath, John J. and Mackay-Sim, Alan (2008) Validation of the comparative quantification method of real-time PCR analysis and a cautionary tale of housekeeping gene selection. Gene Therapy and Molecular Biology, 12 1: 15-24.


Author McCurdy, Richard D.
McGrath, John J.
Mackay-Sim, Alan
Title Validation of the comparative quantification method of real-time PCR analysis and a cautionary tale of housekeeping gene selection
Journal name Gene Therapy and Molecular Biology   Check publisher's open access policy
ISSN 1529-9120
Publication date 2008-01-01
Year available 2008
Sub-type Article (original research)
Volume 12
Issue 1
Start page 15
End page 24
Total pages 10
Place of publication Athens, Greece
Publisher Cancer Therapy
Language eng
Subject 1312 Molecular Biology
1313 Molecular Medicine
Formatted abstract
 Real-time reverse transcription PCR (RT-PCR) is now widely used for quantifying levels of expressed gene transcripts. The present study validates the use of a new RT-PCR analysis method, Comparative Quantification, by comparing it against the ‘gold standard’ Comparative Threshold Cycle method. The former method calculates individual PCR reaction efficiencies, obviating the need for multiple PCRs to generate standard curves from serial dilutions of sample. Real-time reverse transcription PCR was used to verify expression of 18 genes suggested by microarray analysis of schizophrenia versus control fibroblasts. A high correlation (R=0.853) was observed between the two methods, validating Comparative Quantification as a method of RT-PCR data analysis, with the advantage that it is also a quicker and cheaper method. Also, RT-PCR compares the relative expression of target genes to the expression of a reference or “housekeeping” gene in the sample, which is assumed to have stable expression across all samples. Variable expression of the reference gene would reveal itself as a false change in expression in the target gene. The present study investigates the expression of “housekeeping” genes in fibroblast cultures from patients with schizophrenia and matched healthy controls. The results reveal consistent patient versus control differences in expression of commonly used “housekeeping” genes, including GAPDH. We propose that researchers derive housekeeping genes from stable expression data in the system studied and disregard previously published housekeeping genes when designing their real-time PCR experiments.
Keyword Housekeeping gene
Linear regression
Reference gene
Relative quantification
Rotor Gene
Schizophrenia
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
Collection: School of Medicine Publications
 
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