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Abstract
Reverse transcription quantitative PCR (RT-qPCR) is considered today as the gold standard
for accurate, sensitive and fast measurement of gene expression. Unfortunately, what
many users fail to appreciate is that numerous critical issues in the workflow need
to be addressed before biologically meaningful and trustworthy conclusions can be
drawn. Here, we review the entire workflow from the planning and preparation phase,
over the actual real-time PCR cycling experiments to data-analysis and reporting steps.
This process can be captured with the appropriate acronym PCR: plan/prepare, cycle
and report. The key message is that quality assurance and quality control are essential
throughout the entire RT-qPCR workflow; from living cells, over extraction of nucleic
acids, storage, various enzymatic steps such as DNase treatment, reverse transcription
and PCR amplification, to data-analysis and finally reporting.
Copyright 2009 Elsevier Inc. All rights reserved.