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Real Time RT-PCR

Real Time RT-PCR

Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction.
The real-time quantification of the number of amplified products is...

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The choice of reference gene affects statistical efficiency in quantitative PCR data analysis.

Yi Guo¹, Michael L Pennell, Dennis K Pearl

¹Department of Health Outcomes and Policy, College of Medicine, University of Florida, Gainesville, FL.

|October 11, 2013

Summary

Choosing the best reference genes (RGs) for quantitative polymerase chain reaction (qPCR) doesn't always improve statistical efficiency. Our study offers a formula to determine when normalization truly enhances data analysis power.

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Area of Science:

Biomedical research
Molecular biology
Gene expression analysis

Background:

Quantitative polymerase chain reaction (qPCR) is a sensitive technique for measuring gene expression.
Reliable qPCR results depend on normalizing data with stably expressed reference genes (RGs) to control for sample variation.

Purpose of the Study:

To investigate the impact of different reference genes on the statistical efficiency of qPCR data.
To determine if selecting the most stable reference gene guarantees improved data normalization and statistical power.

Main Methods:

Analysis of a quantitative polymerase chain reaction (qPCR) dataset.
Inclusion of 12 target genes and 3 reference genes (RGs).
Evaluation of statistical efficiency and variance based on RG selection.

Main Results:

The most stably expressed reference gene (RG) did not consistently reduce variance or improve statistical efficiency in qPCR data.
Data normalization using RGs does not always enhance statistical efficiency.

Conclusions:

The choice of reference gene significantly impacts the statistical efficiency of quantitative polymerase chain reaction (qPCR) data analysis.
A formula is provided to identify conditions under which reference gene normalization improves statistical efficiency and increases the power of statistical tests.