Good practice guide for the application of quantitative ...

Good practice guide

for the application of

quantitative PCR (qPCR)

Good practice guide for

the application of

quantitative PCR (qPCR)

First Edition 2013

Co-authors

Tania Nolan (1)

Jim Huggett (2)

Elena Sanchez (2)

Contributors

Anders Bergkvist (1)

Malcolm Burns (2)

Rebecca Sanders (2)

Nicholas Redshaw (2)

Tim Wilkes (2)

Acknowledgements

With special thanks to Susan Pang(2) and

Vicki Barwick(2) for their help in the

production of this guide. Acknowledgement

of an individual does not indicate their

agreement with this Guide in its entirety.

Production of this Guide was in part funded

by the UK National Measurement System.

Foreword by Michael Pfaffl

(1)

(2)

(3)

(3)

Sigma-Aldrich

LGC, Teddington

Technical University Of Munich

This publication should be cited as:

T Nolan, J Huggett, E Sanchez, Good practice guide for

the application of quantitative PCR (qPCR), LGC (2013).

Copyright ? 2013 LGC

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Foreword by Michael W Pfaffl

The polymerase chain reaction (PCR) is a rapid, sensitive, and rather simple technique to amplify

DNA, using oligonucleotide primers, dNTPs and a heat stable Taq polymerase. It was invented in

1983 by Kary B. Mullis and co-workers, who, ten years later, were awarded the ¡®Nobel Prize for

Chemistry¡¯. With the introduction of real-time PCR in the late nineties, the PCR method overcame

an important hurdle towards becoming ¡®fully quantitative¡¯ (and therefore known as quantitative

PCR, or qPCR). Currently, qPCR is regarded as the ¡®gold standard¡¯ in the quantitative analysis of

nucleic acids, be it DNA, RNA or micro-RNA molecules. The main reasons for its success are its

high sensitivity, robustness, good reproducibility, broad dynamic quantification range, and very

importantly, affordability. The assay and primer design can often be fully automated and handling

in the lab is blindingly easy.

Another big draw for the user is that, in most instances, the qPCR experiments produce results,

or as we call them, Cq data points. However, the generation of Cq data points is not dependent

on good laboratory practice or the precise application of guidelines such as MIQE. In other words,

when researchers obtain a Cq data point, they need to prove that that particular amplification

result is valid, reliable and meaningful.

And exactly here lies the main challenge of qPCR! This method is ¡®too easy¡¯ to apply and

generates results any time. It is up to the researcher to demonstrate that the data obtained are

valid and if not, investigate where the error could come from.

Hence, it is essential to have a comprehensive understanding of the underlying basic qPCR

principles, sources of error, and general issues inherent to nucleic acid isolation and/or

quantification in order to develop assays and workflows which meet high analytical requirements

in concordance with the MIQE guidelines. Unfortunately, we are still far from having developed

such optimal workflows, with the highest sensitivity or the best RNA integrity metrics, to obtain

reproducible and authentic results.

Thus, I can warmly recommend to the research community this Good Practice Guide for the

Application of Quantitative PCR, with the aim to improve researchers¡¯ experimental workflows,

from sampling to qPCR data analysis, and eventually take us to valid and confident research

results.

Michael W Pfaffl

Michael W. Pfaffl

Physiology Weihenstephan

Technische Universit?t M¨¹nchen

Weihenstephaner Berg 3

85354 Freising

Germany

Michael.Pfaffl@wzw.tum.de

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Good Practice Guide for the Application of Quantitative

PCR (qPCR)

Contents

Objectives of this guide ....................................................................................................................... 2

Terminology .......................................................................................................................................... 3

Common abbreviations ........................................................................................................................ 7

Section A. Technical information ....................................................................................................... 8

A.1

PCR and qPCR ......................................................................................................................... 8

A.2

Sample purification and Quality Control (QC) .................................................................... 10

A.3

qPCR assay design ............................................................................................................... 21

A.4

Assay optimisation and validation ...................................................................................... 32

A.5

Normalisation ......................................................................................................................... 41

A.6

Data analysis .......................................................................................................................... 44

A.7

Troubleshooting .................................................................................................................... 50

Section B. Applications of qPCR ...................................................................................................... 53

B.1

Use of PCR and qPCR in food authenticity studies ........................................................... 53

B.2

Pathogen detection for clinical analysis ............................................................................. 59

B.3

MicroRNA expression profiling ............................................................................................ 61

Section C: Protocols .......................................................................................................................... 64

C.1

Basic qPCR protocol ............................................................................................................. 65

C.2

The SPUD assay for detection of assay inhibitors ............................................................ 68

C.3

3¡¯/5¡¯ Assay for analysis of template integrity ..................................................................... 70

C.4

Reverse transcription (one-step and two-step) protocols ................................................ 72

C.5

Primer optimisation ............................................................................................................... 78

C.6

qPCR reference gene selection protocol ............................................................................ 84

C.7

qPCR efficiency determination protocol ............................................................................. 87

C.8

qPCR gene expression analysis protocols......................................................................... 90

Section D: Further resources ............................................................................................................ 94

Section E: Further reading ................................................................................................................ 95

Section F: References ........................................................................................................................ 96

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Objectives of this guide

There is little doubt that PCR (Polymerase Chain Reaction) has transformed the fields of clinical

and biological research, due to its robustness and simplicity. Subsequent developments, such as

real-time quantitative PCR (qPCR) and reverse transcription qPCR (RT-qPCR), offer simple

methods for analysis of DNA and RNA molecules. However, completing qPCR assays to a high

standard of analytical quality can be challenging for a number of reasons, which are discussed in

detail in this guide.

qPCR has a large number of applications in a wide range of areas, including healthcare and food

safety. It is therefore of paramount importance that the results obtained are reliable in themselves

and comparable across different laboratories.

This guide is aimed at individuals who are starting to use qPCR and realise that, while this

method is easy to perform in the laboratory, numerous factors must be considered to ensure that

the method will be applied correctly.

These additional considerations include ¨C but are not limited to ¨C methods of sampling, sample

storage, nucleic acid extraction, and nucleic acid storage, manipulation and preparation. In other

words, all the steps prior to undertaking the quantification technique must also be controlled. At

the other end of the analytical process, reporting technical results may be highly subjective. Since

qPCR is a relative method, requiring the comparison of two or more samples to a standard curve

or to each other, standardisation of results is very challenging. The primary qPCR metric, the

quantification cycle (Cq)1, depends on many factors including where a threshold is set, the choice

of reporter and day-to-day variation in measurement. In addition, since Cq exists on a logarithmic

scale, there are specific statistical challenges that need to be addressed to analyse these data

accurately.

All these factors combine to make a technically simple technique, challenging to interpret with

absolute confidence. This guide aims to assist those who are, or will be, using qPCR by

discussing the issues that need consideration during experimental design. The guide entails ¡°tried

and tested¡± approaches, and troubleshoots common issues.

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