Editing Reverse transcription polymerase chain reaction (section)

== Publication guidelines ==

Quantitative  RT-PCR assay is considered to be the gold standard for measuring the number of copies of specific cDNA targets in a sample but it is poorly standardized.<ref name="urlwww.microarrays.ca">{{cite web |url=http://www.microarrays.ca/about/reviews/Bustin_Review_July2009.pdf |title=www.microarrays.ca }}</ref> As a result, while there are numerous publications utilizing the technique, many provide inadequate experimental detail and use unsuitable data analysis to draw inappropriate conclusions. Due to the inherent variability in the quality of any quantitative PCR data, not only do reviewers have a difficult time evaluating these manuscripts, but the studies also become impossible to replicate.<ref>{{cite journal |author=Bustin SA |title=Why the need for qPCR publication guidelines?--The case for MIQE |journal=Methods |volume=50 |issue=4 |pages=217–26 |date=April 2010 |pmid=20025972 |doi=10.1016/j.ymeth.2009.12.006 }}</ref> Recognizing the need for the standardization of the reporting of experimental conditions, the [[Minimum Information for Publication of Quantitative Real-Time PCR Experiments]] (MIQE, pronounced mykee) guidelines have been published by an international consortium of academic scientists. The MIQE guidelines describe the minimum information necessary for evaluating [[quantitative PCR]] experiments that should be required for publication to encourage better experimental practice and ensuring the relevance, accuracy, correct interpretation, and repeatability of quantitative PCR data.<ref name="Bustin et al">{{cite journal  |vauthors=Bustin SA, Benes V, Garson JA, etal |title=The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments |journal=Clin. Chem. |volume=55 |issue=4 |pages=611–22 |date=April 2009 |pmid=19246619 |doi=10.1373/clinchem.2008.112797 |doi-access=free }}</ref>

Besides reporting guidelines, the MIQE stresses the need to standardize the nomenclature associated with quantitative PCR to avoid confusion; for example, the abbreviation ''qPCR'' should be used for [[Real-time polymerase chain reaction|quantitative real-time PCR]], while ''RT-qPCR'' should be used for reverse transcription-qPCR, and genes used for normalization should be referred to as ''reference genes'' instead of ''[[housekeeping gene]]s''. It also proposes that commercially derived terms like ''TaqMan probes'' should not be used, but instead referred to as ''[[hydrolysis probes]]''. Additionally, it is proposed that the quantification cycle (Cq) be used to describe the PCR cycle used for quantification instead of the threshold cycle (Ct), crossing point (Cp), and takeoff point (TOP), which refer to the same value but were coined by different manufacturers of [[Quantitative PCR instrument|real-time instruments]].<ref name="urlwww.microarrays.ca"/>

The guideline consists of the following elements: 1) experimental design, 2) sample, 3) nucleic acid extraction, 4) reverse transcription, 5) qPCR target information, 6) oligonucleotides, 7) protocol, 8) validation, and 9) data analysis. Specific items within each element carry a label of either E (essential) or D (desirable). Those labeled E are considered critical and indispensable while those labeled D are considered peripheral yet important for best practices.<ref name="Bustin et al"/>