Quantification of mRNA using real-time RT-PCR

  title={Quantification of mRNA using real-time RT-PCR},
  author={Tania Nolan and Rebecca E Hands and Stephen A Bustin},
  journal={Nature Protocols},
The real-time reverse transcription polymerase chain reaction (RT-qPCR) addresses the evident requirement for quantitative data analysis in molecular medicine, biotechnology, microbiology and diagnostics and has become the method of choice for the quantification of mRNA. Although it is often described as a “gold” standard, it is far from being a standard assay. The significant problems caused by variability of RNA templates, assay designs and protocols, as well as inappropriate data… 

The use of real-time quantitative PCR for the analysis of cytokine mRNA levels.

This chapter focuses on the details of RNA isolation and cDNA synthesis methods, on the application of RT-qPCR for measurements of cytokine mRNA levels using Sybr-Green I as detection chemistry, and the pros and contras of the absolute quantification versus relative quantification analysis.

Relative quantification

While real-time RT-PCR has a tremendous potential for analytical and quantitative applications in transcriptome analysis, a comprehensive understanding of its underlying quantification principles is important.

Quantitative Real-Time RT-PCR

The first part of this chapter provides a detailed description of the most commonly used real-time-based chemistries, SybrGreen and TaqMan, focusing especially on their advantages when applied in FFPE material, and general guidelines that should be followed to optimize the design of quantitative real- time PCR assays.

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This study demonstrates that the variability of the RT step is sufficiently large to call into question the validity of many published data that rely on quantification of cDNA.

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The concept of normalization in transcript quantification is introduced here in an attempt to convince molecular biologists, and non-specialists, that systematic validation of reference genes is essential for producing accurate, reliable data in qRT-PCR analyses, and thus should be an integral component of them.

Evaluation of Quantitative RT-PCR Using Nonamplified and Amplified RNA

1 round of linear RNA amplification, even with suboptimal-quality RNA, is appropriate to generate reproducible and high-fidelity qRT-PCR relative expression data that have similar confidence levels as those from NA samples.

The importance of selecting the appropriate reference genes for quantitative real time PCR as illustrated using colon cancer cells and tissue

There is a variation in the fold changes obtained dependent on the number of reference genes used, which demonstrates the need for careful experimental design in RT-qPCR studies to help eliminate false interpretation and reporting of results.

Design and optimization of reverse-transcription quantitative PCR experiments.

It is shown how an optimal sampling plan can be calculated for a limited budget and the use of sample replicates preferentially to any other replicates when working with solid tissue, cell cultures, and single cells is recommended.

Setting up reverse transcription quantitative-PCR experiments.

A qRT-PCR protocol is described that illustrates the essential technical steps required to generate quantitative data that are reliable and reproducible and suitable for the validation of weakly expressed genes (≥0.5 fold), identified through microarray analysis.



Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays.

  • S. Bustin
  • Biology
    Journal of molecular endocrinology
  • 2000
The technical aspects involved are discussed, conventional and kinetic RT-PCR methods for quantitating gene expression are contrasted, and the usefulness of these assays are illustrated by demonstrating the significantly different levels of transcription between individuals of the housekeeping gene family, glyceraldehyde-3-phosphate-dehydrogenase (GAPDH).

Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems.

  • S. Bustin
  • Biology
    Journal of molecular endocrinology
  • 2002
A snapshot of the state-of-the-art in real-time RT-PCR is provided and some of the problems associated with interpreting results that are numerical and lend themselves to statistical analysis, yet whose accuracy is significantly affected by reagent and operator variability are described.

Pitfalls of quantitative real-time reverse-transcription polymerase chain reaction.

Real-time RT-PCR remains a research tool, and it is important to recognize the considerable pitfalls associated with transcriptome analysis, with the successful application of RTPCR depending on careful experimental design, application, and validation.

RNA integrity and the effect on the real-time qRT-PCR performance.

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Although it is evident that qRT-PCR assay has become a useful and important technology in the clinical diagnostic laboratory, it must be used appropriately and it is essential to be aware of its limitations if it is to fulfil its potential.

Properties of the reverse transcription reaction in mRNA quantification.

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

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

Real-time, fluorescence-based quantitative PCR: a snapshot of current procedures and preferences

  • S. Bustin
  • Biology
    Expert review of molecular diagnostics
  • 2005
The survey reveals extensive interlaboratory variation in assay design, validation and analysis that, together with other dubious practices, are the likely cause for the publication of variable results of real-time reverse transcription PCR results.