Are MIQE Guidelines Being Adhered to in qPCR Investigations in Photobiomodulation Experiments?

The polymerase chain reaction (PCR) is an important and reliable technology for research and diagnostic analysis, and is a quick and easy method of enzymatically synthesizing and amplifying unlimited copies of specific DNA sequences in a few short hours. From its inception, PCR has matured over the years from a laborious, timeconsuming, and gel-based technique to an automated, high throughput, rapid quantitative technique. This technique, which formed the cornerstone of the human genome project, was only developed 20 years ago. The technique as we know it originates from research conducted in the 1980s at Cetus Corporation in California. The story begins in 1983 with Kary Mullis, PhD, who came up with the idea while driving one day. The novelty and potential impact of the idea was developed and the theory became a reality. In 1985, the technology was presented for the first time and the first set of results was published later that year in Science. In 1993, Mullis was awarded the Nobel Prize for chemistry. Initially, the technique was slow and arduous, and required the manual transfer of tubes between water baths set at different temperatures, and the DNA polymerase first used (isolated from Escherichia coli) was inactivated during DNA denaturation and had to be manually replaced at the start of each cycle. As the years passed, the technology developed and significant advances were made with the discovery of Taq polymerase (isolated from Thermus aquaticus), which was able to withstand the high temperatures required during DNA denaturation, and the development of the closed thermal cycler by PerkinElmer. These developments have led to the streamlining and shortening of the process, and minimizing the number of steps that require human interaction. Today, the technology has evolved into a quantitative, fluorescence-based real-time, quantitative PCR (qPCR). qPCR is currently the gold standard and global mainstay for the quantification of microRNA and messenger RNA (mRNA); however, there is a downside to the technique that is related to the adaptations to the methodology and inconsistent publication of technical information. As a consequence, qPCR can become an inadequately standardized and inconsistent technique. The lack of agreement on how best to perform and interpret qPCR experiments and insufficient experimental detail led to the development of the minimum information for publication of quantitative real-time PCR experiments (MIQE) guidelines in 2009, which is aimed at ensuring integrity and reliability of publications, promote consistency between different research groups, and increase experimental transparency. MIQE is a set of guidelines describing the minimum information that should be reported on when publishing qPCR experiments. These guidelines cover aspects related to sample acquisition, experimental design and validation, and data analysis. These guidelines are easily accessible and available from the MIQE gene-quantification info website (http://miqe.gene-quantification.info). Three main causes for variable qPCR results have been identified, and include biological variability, technical variability, and experimental design. Biological variability is more difficult to control, and is determined by natural genotypic and phenotypic variation between individuals, cells, and tissues. qPCR is dependent on the organism, tissue type, and time of experiment (time of extraction of nucleic acid post-treatment), and thus conclusions should be placed into context. Technical aspects that affect qPCR performance and variability include sample type, isolation, storage, handling, and preparation; replicate numbers (biological versus technical replicates); nucleic acid quality, quantity, and purity; choice of reverse transcription (RT) primers and probes; assay design; methods of normalization; and data and statistical analysis. These technical aspects are all in the hands of the researchers, and the numerous steps involved in qPCR allow for the introduction of assay discrepancies and errors. When it comes to reporting on assay optimization, one should include database accession numbers (as there can be variants for the same gene), amplicon size, primer sequence, and probe sequence (including any modified bases), position, and dye linkage. A problem with supplying this information is introduced by companies who supply ready-made primers, probes, and assays. For self-designed primers, the design software should be stated and primer specificity (using BLAST) should be validated. Primer optimization, including MgCl2 concentration and annealing temperature, and priming conditions should be provided. There should also be evidence of intraand interassay precision, which are measures of repeatability and reproducibility, respectively.