Properties of targeted preamplification in DNA and cDNA quantification

Abstract

Objective: Quantification of small molecule numbers often requires preamplification to generate enough copies for accurate downstream enumerations. Here, we studied experimental parameters in targeted preamplification and their effects on downstream quantitative real-time PCR (qPCR). Methods: To evaluate different strategies, we monitored the preamplification reaction in real-time using SYBR Green detection chemistry followed by melting curve analysis. Furthermore, individual targets were evaluated by qPCR. Result: The preamplification reaction performed best when a large number of primer pairs was included in the primer pool. In addition, preamplification efficiency, reproducibility and specificity were found to depend on the number of template molecules present, primer concentration, annealing time and annealing temperature. The amount of nonspecific PCR products could also be reduced about 1000-fold using bovine serum albumin, glycerol and formamide in the preamplification. Conclusion: On the basis of our findings, we provide recommendations how to perform robust and highly accurate targeted preamplification in combination with qPCR or next-generation sequencing.

Keywords:

• experimental design
• multiplex PCR
• preamplification
• primer-pools
• quantitative real-time PCR
• single-cell analysis
• targeted preamplification

Acknowledgements

The authors are grateful to S Dolatabadi for primer design and to S Busch for scientific discussion of data.

Financial & competing interests disclosure

A Ståhlberg is supported by the Assar Gabrielssons Research Foundation, LUA/ALF Västra Götaland, Johan Jansson Foundation for Cancer Research, Swedish Cancer Society, Swedish Society for Medical Research, Swedish Research Council, Swedish Society for Medicine, BioCARE National Strategic Research Program at University of Gothenburg, VINNOVA, Wilhelm and Martina Lundgren Foundation for Scientific Research and Åke Winbergs Foundation. A Ståhlberg is a shareholder of the TATAA Biocenter. M Kubista is supported by grants ERDF (LK21305 and BIOCEV CZ.1.05/1.1.00/02.0109), Ministry of Youth, Education and Sports of the Czech Republic (AV0Z50520701). M Kubista is a shareholder and employee of the TATAA Biocenter and MultiD Analyses. G Landberg is supported by the Swedish Research Council, BioCARE National Strategic Research Program at University of Gothenburg, VINNOVA and Swedish Cancer Society. D Svec is supported by the Academy of Science, Czech Republic. D Svec is a shareholder of the TATAA Biocenter. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Key issues

• Analyses of limited sample sizes, including rare tissues, liquid biopsies, fine-needle aspirates and single cells are becoming important in numerous research and clinical assessments.

• Quantification of small numbers of DNA and RNA molecules usually requires preamplification for accurate analysis by downstream qPCR or next-generation sequencing.

• The most common strategy for targeted preamplification is based on multiplex PCR using pools of specific primer pairs, but the properties of the reaction are poorly understood.

• The overall performance of target preamplification can be studied in real-time, using SYBR Green I detection chemistry followed by melting curve analysis.

• The applied number of preamplification cycles should be sufficient to produce at least five (accurate sensitivity), but preferably 35 (accurate precision), molecules per aliquot used in downstream qPCR.

• The amount of initial template molecules, number of assays used, primer concentration, annealing time and annealing temperature are key parameters that influence the sensitivity, specificity, efficiency and reproducibility of targeted preamplification.

• Addition of additives, including bovine serum albumin in combination with glycerol or formamide, may improve preamplification by reducing the formation of nonspecific PCR products.

• Single-cell gene expression profiling is feasible using qPCR, applying carefully optimized targeted preamplification.

• On the basis of experimental data, we are able to provide guidelines how to perform accurate targeted preamplification for downstream applications.

Notes