Inappropriate selection of reference genes and degraded RNA can lead to flawed data and erroneous results in reverse transcription quantitative PCR (RT-qPCR) studies. Scientists at INRS-Institut Armand Frappier in Laval, Quebec, in collaboration with Bio-Rad Laboratories, Inc., came to these conclusions in a paper published recently in Molecular Biotechnology.
New Study Demonstrates False Conclusions Reached if MIQE Not Followed in Human Placenta Quantitative PCR Studies
RT-qPCR is routinely used to determine gene expression differences among a wide variety of samples. However, errors in sample processing and handling and primer design and validation, as well as reference gene selection, can result in erroneous experimental conclusions. The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines were developed in 2009 to establish baseline information requirements for each of these steps to ensure that qPCR data published in articles are accurate and reproducible. Clinical Chemistry, Nucleic Acid Research, and BioMed Central now require qPCR papers to adhere to MIQE guidelines in order to be published.
"This paper demonstrates very clearly how poor sample quality and inappropriate data analysis can distort qPCR results,” said Stephen Bustin, first author of the Clinical Chemistry paper that introduced MIQE. “The paper also illustrates beautifully how straightforward it is to follow the MIQE guidelines and how the detailed reporting of the various parameters helps the reader comprehend the protocol and have confidence in the results.”
When two students in the lab of Cathy Vaillancourt, an INRS-Institut Armand Frappier professor, obtained different results doing the same experiment, Vaillancourt set out to understand what introduces variability into RT-qPCR experiments and what her lab could do to prevent it.
After attending a presentation demonstrating the impact of RNA integrity and reference gene selection on the expression analysis of a breast cancer protein by Bio-Rad field application specialist Sean Taylor, Vaillancourt reached out to Sean. They collaborated to investigate the same parameters on RT-qPCR studies of a gene (OGG1) that is known to be regulated in human placenta tissues and whose expression is complicated by diabetes and preeclampsia.
“Sean’s MIQE studies in the gene expression analysis of a cancer protein inspired us to test his conclusions on OGG1 expression in placental tissue,” said Vaillancourt. “The study shows not only how RNA quality and appropriate reference gene selection is important to obtain accurate and reproducible RT-qPCR data, but how different and even opposite results can be reported if the key steps in the MIQE guidelines are not followed.”
Importance of RNA Integrity
The first step in RT-qPCR is the extraction of total RNA from samples. Degraded RNA can have a significant impact on RT-qPCR data and is a particularly serious issue in human placental tissue due to variable sample storage conditions and high levels of RNAse.
The authors found that OGG1 expression was lower in degraded RNA compared to intact samples and that the significant difference in normalized expression between tissue from normal and preeclamptic pregnancies was lost.
According to the authors of the study, “These data have widespread implications for the analysis of the many types of human tissue that are sensitive to degradation for which sample handling and storage is inconsistent.”
RNA samples were analyzed using Bio-Rad’s Experion™ automated electrophoresis system. Samples receiving an RNA Quality Indicator score greater than 8 were designated as intact RNA. About 20% of placenta tissue RNA samples in the study were significantly degraded, with RQI values between 3 and 5.
Selecting Stable Reference Genes
According to the MIQE website, a high proportion of papers still report expression patterns of target genes normalized against a single, nonvalidated reference gene. To illustrate the significance of selecting appropriate reference genes to normalize target gene expression, the normalized expression of OGG1 was analyzed in normotensive (normal blood pressure) versus preeclamptic pregnancy placentas. Normalization with the stable genes yielded a significant increase of OGG1 expression in preeclamptic compared to normotensive pregnancy placentas, as expected according to the literature. However, normalization with unstable genes, such as the 18S rRNA gene, led to no significant difference in OGG1 expression or even the opposite of what was predicted.
To identify stable reference genes, the researchers characterized nine of the most commonly used, including the “usual suspects,” whose stability in experimental conditions is not typically tested: 18S rRNA, GADPH, and beta-actin The stability of these expressed genes in pathological pregnancy conditions was statistically validated using software tools geNorm and NormFinder.
Bio-Rad is dedicated to promoting the integrity of qPCR research and offers an array of technology and resources to help researchers become MIQE-compliant, including integrated reference gene stability measures in CFX Manager™ software and the Bio-Rad sponsored MIQE qPCR iPad app. To learn more, watch a MIQE tutorial at http://bit.ly/MIQE_Video. Scientists can also read a paper exploring the impact of MIQE parameters in plant tissue.
“Bio-Rad has been amongst the earliest supporters of the MIQE guidelines and it is encouraging to see that their investment in educating the research community has resulted in the publication of this paper,” Bustin said.