Abstract
Reverse transcription quantitative PCR is an established, simple and effective method for RNA measurement. However, technical standardisation challenges combined with frequent insufficient experimental detail render replication of many published findings challenging. Consequently, without adequate consideration of experimental standardisation, such findings may be sufficient for a given publication but cannot be translated to wider clinical application. This article builds on earlier standardisation work and the MIQE guidelines, discussing processes that need consideration for accurate, reproducible analysis when dealing with patient samples. By applying considerations common to the science of measurement (metrology), one can maximise the impact of gene expression studies, increasing the likelihood of their translation to clinical tools. ᅟ
Highlights
The real-time quantitative polymerase chain reaction [1], developed from the revolutionary method of polymerase chain reaction (PCR) pioneered by Kary Mullis in the 1980s [2,3,4], has emerged as a widely used method for biological investigation because it can detect and precisely quantify very small amounts of specific nucleic acid sequences. This is coupled to an inherent simplicity that makes qPCR assays straightforward to design and perform
The characterisation of gene expression patterns through quantification of messenger RNA, by coupling reverse transcription with PCR, as a surrogate of cell metabolism is a major application of this technology
Fundamental experimental details are often omitted when reporting gene expression measurements, including information pertaining to RNA quality, the rationale for the choice of the normalisation strategy, the location of the amplicon or detailed descriptions of the reverse transcriptase and PCR assay conditions [22, 23]
Summary
The real-time quantitative polymerase chain reaction (qPCR) [1], developed from the revolutionary method of polymerase chain reaction (PCR) pioneered by Kary Mullis in the 1980s [2,3,4], has emerged as a widely used method for biological investigation because it can detect and precisely quantify very small amounts of specific nucleic acid sequences. RT-qPCR has been used to measure bacterial gene expression [7, 8] or RNA viral loads [9,10,11,12], to evaluate cancer status or to track disease progression and response to treatment [13,14,15]. Fundamental experimental details are often omitted when reporting gene expression measurements, including information pertaining to RNA quality, the rationale for the choice of the normalisation strategy, the location of the amplicon or detailed descriptions of the reverse transcriptase and PCR assay conditions [22, 23].
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
