A simple, accurate and universal method for quantification of PCR

Research into gene expression enables scientists to decipher the complex regulatory networks that control fundamental biological processes. Quantitative real-time PCR (qPCR) is a powerful and ubiquitous method for interrogation of gene expression. Accurate quantification is essential for correct int...

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Bibliographic Details
Published in:BMC biotechnology 2016-03, Vol.16 (27), p.27-27, Article 27
Main Authors: Boulter, Nicky, Suarez, Francia Garces, Schibeci, Stephen, Sunderland, Trevor, Tolhurst, Ornella, Hunter, Tegan, Hodge, George, Handelsman, David, Simanainen, Ulla, Hendriks, Edward, Duggan, Karen
Format: Article
Language:English
Subjects:
Analysis
Animals
Calibration
Cells, Cultured
DNA - analysis
DNA - genetics
Dyes
Efficiency
Gene Expression
Humans
Laboratories
Leukocytes, Mononuclear
Methodology
Methods
Mice
Performance evaluation
Polymerase chain reaction
Real-Time Polymerase Chain Reaction - methods
Real-Time Polymerase Chain Reaction - standards
RNA
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Summary:Research into gene expression enables scientists to decipher the complex regulatory networks that control fundamental biological processes. Quantitative real-time PCR (qPCR) is a powerful and ubiquitous method for interrogation of gene expression. Accurate quantification is essential for correct interpretation of qPCR data. However, conventional relative and absolute quantification methodologies often give erroneous results or are laborious to perform. To overcome these failings, we developed an accurate, simple to use, universal calibrator, AccuCal. Herein, we show that AccuCal quantification can be used with either dye- or probe-based detection methods and is accurate over a dynamic range of ≥10(5) copies, for amplicons up to 500 base pairs (bp). By providing absolute quantification of all genes of interest, AccuCal exposes, and circumvents, the well-known biases of qPCR, thus allowing objective experimental conclusions to be drawn. We propose that AccuCal supersedes the traditional quantification methods of PCR.
ISSN:1472-6750
1472-6750
DOI:10.1186/s12896-016-0256-y