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      Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes

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          Abstract

          Using real-time reverse transcription PCR ten housekeeping genes from different abundance and functional classes in various human tissues were evaluated. The conventional use of a single gene for normalization leads to relatively large errors in a significant proportion of samples tested.

          Abstract

          Background

          Gene-expression analysis is increasingly important in biological research, with real-time reverse transcription PCR (RT-PCR) becoming the method of choice for high-throughput and accurate expression profiling of selected genes. Given the increased sensitivity, reproducibility and large dynamic range of this methodology, the requirements for a proper internal control gene for normalization have become increasingly stringent. Although housekeeping gene expression has been reported to vary considerably, no systematic survey has properly determined the errors related to the common practice of using only one control gene, nor presented an adequate way of working around this problem.

          Results

          We outline a robust and innovative strategy to identify the most stably expressed control genes in a given set of tissues, and to determine the minimum number of genes required to calculate a reliable normalization factor. We have evaluated ten housekeeping genes from different abundance and functional classes in various human tissues, and demonstrated that the conventional use of a single gene for normalization leads to relatively large errors in a significant proportion of samples tested. The geometric mean of multiple carefully selected housekeeping genes was validated as an accurate normalization factor by analyzing publicly available microarray data.

          Conclusions

          The normalization strategy presented here is a prerequisite for accurate RT-PCR expression profiling, which, among other things, opens up the possibility of studying the biological relevance of small expression differences.

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          Most cited references 24

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          Quantitative monitoring of gene expression patterns with a complementary DNA microarray.

          A high-capacity system was developed to monitor the expression of many genes in parallel. Microarrays prepared by high-speed robotic printing of complementary DNAs on glass were used for quantitative expression measurements of the corresponding genes. Because of the small format and high density of the arrays, hybridization volumes of 2 microliters could be used that enabled detection of rare transcripts in probe mixtures derived from 2 micrograms of total cellular messenger RNA. Differential expression measurements of 45 Arabidopsis genes were made by means of simultaneous, two-color fluorescence hybridization.
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            Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays.

             SA Bustin (2000)
            The reverse transcription polymerase chain reaction (RT-PCR) is the most sensitive method for the detection of low-abundance mRNA, often obtained from limited tissue samples. However, it is a complex technique, there are substantial problems associated with its true sensitivity, reproducibility and specificity and, as a quantitative method, it suffers from the problems inherent in PCR. The recent introduction of fluorescence-based kinetic RT-PCR procedures significantly simplifies the process of producing reproducible quantification of mRNAs and promises to overcome these limitations. Nevertheless, their successful application depends on a clear understanding of the practical problems, and careful experimental design, application and validation remain essential for accurate quantitative measurements of transcription. This review discusses the technical aspects involved, contrasts conventional and kinetic RT-PCR methods for quantitating gene expression and compares the different kinetic RT-PCR systems. It illustrates the usefulness of these assays by demonstrating the significantly different levels of transcription between individuals of the housekeeping gene family, glyceraldehyde-3-phosphate-dehydrogenase (GAPDH).
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              Real time quantitative PCR.

              We have developed a novel "real time" quantitative PCR method. The method measures PCR product accumulation through a dual-labeled fluorogenic probe (i.e., TaqMan Probe). This method provides very accurate and reproducible quantitation of gene copies. Unlike other quantitative PCR methods, real-time PCR does not require post-PCR sample handling, preventing potential PCR product carry-over contamination and resulting in much faster and higher throughput assays. The real-time PCR method has a very large dynamic range of starting target molecule determination (at least five orders of magnitude). Real-time quantitative PCR is extremely accurate and less labor-intensive than current quantitative PCR methods.
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                Author and article information

                Journal
                Genome Biol
                Genome Biology
                BioMed Central (London )
                1465-6906
                1465-6914
                2002
                18 June 2002
                : 3
                : 7
                : research0034.1-research0034.11
                Affiliations
                [1 ]Center for Medical Genetics, Ghent University Hospital 1K5, De Pintelaan 185, B-9000 Ghent, Belgium
                Corresondence: Frank Speleman. E-mail: franki.speleman@rug.ac.be
                gb-2002-3-7-research0034
                126239
                12184808
                Copyright © 2002 Vandesompele et al., licensee BioMed Central Ltd
                Categories
                Research

                Genetics

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