The State of RT-Quantitative PCR: Firsthand Observations of Implementation of Minimum Information for the Publication of Quantitative Real-Time PCR Experiments (MIQE)

Abstract

In the past decade, the techniques of quantitative PCR (qPCR) and reverse transcription (RT)-qPCR have become accessible to virtually all research labs, producing valuable data for peer-reviewed publications and supporting exciting research conclusions. However, the experimental design and validation processes applied to the associated projects are the result of historical biases adopted by individual labs that have evolved and changed since the inception of the techniques and associated technologies. This has resulted in wide variability in the quality, reproducibility and interpretability of published data as a direct result of how each lab has designed their RT-qPCR experiments. The ‘minimum information for the publication of quantitative real-time PCR experiments' (MIQE) was published to provide the scientific community with a consistent workflow and key considerations to perform qPCR experiments. We use specific examples to highlight the serious negative ramifications for data quality when the MIQE guidelines are not applied and include a summary of good and poor practices for RT-qPCR.

Gut microbiota related to Giardia duodenalis, Entamoeba spp. and Blastocystis hominis infections in humans from Côte d’Ivoire

“…Standard curves for each bacterial species/group were done using a range of 10 9 -10 10 copies of the plasmid (pUC57) containing one copy of the target [21]. The qPCR runs were analyzed using the 7300 System SDS Software (Applied Biosystems, Foster City, USA), and results expressed followed the minimum information for publication of quantitative real-time PCR experiments (MIQE) guidelines [22,23].…”

Selection of housekeeping genes for quantitative gene expression analysis in yellow-feathered broilers

“…The MIQE principle and operational method to determine gene expression were used in this paper (Bustin et al 2009;Taylor and Mrkusich 2014;Gao et al 2016). Total tissue RNA was extracted using RNAprep Pure Tissue Kit (DP431, Beijing Tiangen Biotechnology Co. Ltd., China) from liver and jejunum of chickens according to the manufacturer's instructions.…”

Técnica De Reacción De Polimerasa en Cadena (Qpcr) en Tiempo Real Para La Identificación Y Cuantificación De Streptococcus Mutans en Saliva Y Biopelícula Dentaria De Niños

“…24,[30][31] On the other hand, this improved technique of conventional PCR, which is a qualitative test, allows conducting qualitative and quantitative measurements of specific genes in a sample of nucleic acid. An example of this is SYBR-Green, a fluorometric detection method designed to amplify a DNA sequence not exceeding 250 pb, donde el reactivo se une inespecíficamente a la doble hebra de DNA produciendo fluorescencia cuantitativa proporcional al número de moléculas iniciales, 29,[32][33][34][35] lo que da como resultado una cuantificación exacta, sensible y rápida.…”

“…where the reagent non-specifically binds to a double DNA strand producing quantitative fluorescence proportional to the initial number of molecules, 29,[32][33][34][35] resulting in an accurate, sensitive, and fast quantification.…”

“…Con base en el tamaño del genoma de S. mutans se calculó el número de bacterias en mL de saliva y biopelícula dental. 35 The values obtained by quantifying DNA of S. mutans in saliva and dental biofilm samples by qPCR are shown in table 4. The number of bacteria in mL of saliva and dental biofilm was calculated based on the size of the S mutans genome.…”