The ongoing evolution of qPCR

The polymerase chain reaction (PCR) is usually described as a simple, sensitive and rapid technique that uses oligonucleotide primers, dNTPs and a heat stable Taq polymerase to amplify DNA. It was invented by Mullis and co-workers [1,2] in the early eighties, who were awarded the 1993 Nobel Prize for chemistry for this discovery. With the discovery of real-time PCR in the nineties the method took an important hurdle towards becoming ‘‘fully quantitative” [3]. The addition of an initial reverse transcription (RT) step produced the complementary RT-PCR, a powerful means of amplifying any type of RNA [4,5]. Today quantitative PCR (qPCR) is widely used in research and diagnostics, with numerous scientists contributing to the pre-eminence of PCR in a huge range of DNA-, RNA(coding and non-coding) or protein(immunoor proximity ligation assay qPCR) based applications. Soon the PCR was regarded as the ‘‘gold standard” in the quantitative analysis of nucleic acid, because of its high sensitivity, good reproducibility, broad dynamic quantification range, easy use and reasonable good value for money [6–8]. qPCR has substantial advantages in quantifying low target copy numbers from limited amounts of tissue or identifying minor changes in mRNA or microRNA expression levels in samples with low RNA concentrations or from single cells analysis [9–11]. The extensive potential to quantify nucleic acids in any kind of biological matrix has kept qPCR at the forefront of extensive research efforts aimed at developing new or improved applications. But are qPCR and its associated quantification workflow really as simple as we assume? It is essential to have a comprehensive understanding of the underlying basic principles, error sources and general problems inherent with qPCR and RT-qPCR. This rapidly reveals the urgent need to promote efforts towards more reproducible, sensitive, truly quantitative and, ultimately, more biologically valid experimental approaches. Therefore, the challenge is to develop assays that meet current analytical requirements and anticipate new problems, for example in novel biological matrices or for higher throughput applications. Unfortunately, we are far from having developed optimal workflows, the highest sensitivity, the best RNA integrity metrics or the ultimate real-time cycler, all of which are indispensable for optimal PCR amplification and authentic results. The qPCR research community still aims to improve and evolve, which brings to the topic of this PCR special issue – The ongoing evolution of qPCR. In this issue we want to focus on some selected application fields which have been identified as indispensable for research and diagnostics: Standardisation – Why do we need more standardisation and therefore the MIQE (minimum information for publication of quan-