Endogenous control genes in complex vascular tissue samples.
BACKGROUND
Gene expression microarrays and real-time PCR are common methods used to measure mRNA levels. Each method has a fundamentally different approach of normalization between samples. Relative quantification of gene expression using real-time PCR is often done using the 2(/\)(-DeltaDeltaCt) method, in which the normalization is performed using one or more endogenous control genes. The choice of endogenous control gene is often arbitrary or bound by tradition. We here present an analysis of the differences in expression results obtained with microarray and real-time PCR, dependent on different choices of endogenous control genes.


RESULTS
In complex tissue, microarray data and real-time PCR data show the best correlation when endogenous control genes are omitted and the normalization is done relative to total RNA mass, as measured before reverse transcription.


CONCLUSIONS
We have found that for real-time PCR in heterogeneous tissue samples, it may be a better choice to normalize real-time PCR Ct values to the carefully measured mass of total RNA than to use endogenous control genes. We base this conclusion on the fact that total RNA mass normalization of real-time PCR data shows better correlation to microarray data. Because microarray data use a different normalization approach based on a larger part of the transcriptome, we conclude that omitting endogenous control genes will give measurements more in accordance with actual concentrations.
Folkersen
Lasse
L
Cardiovasular Research Unit, Center for Molecular Medicine, Department of Medicine, Karolinska Institute, Stockholm, Sweden.
Kurtovic
Sanela
S
Razuvaev
Anton
A
Agardh
Hanna E
HE
Gabrielsen
Anders
A
Paulsson-Berne
Gabrielle
G
D000368:Aged; D001808:Blood Vessels / Q000166:cytology* / Q000378:metabolism* / Q000473:pathology; D016893:Carotid Stenosis / Q000235:genetics / Q000473:pathology; D042783:Endothelial Cells / Q000378:metabolism; D005260:Female; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D006801:Humans; D007962:Leukocytes / Q000378:metabolism; D008297:Male; D032389:Myocytes, Smooth Muscle / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D019914:Taq Polymerase / Q000378:metabolism; D013997:Time Factors
D019914:Taq Polymerase