title: Quantitative PCR pitfalls: the case of the human placenta.
abstract: Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) is a rapid and high throughput gene expression quantification technology. In order to obtain accurate results, several key experimental design and standardization steps must be rigorously followed as previously described in the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines. This study investigates the effect of reference gene normalization and the impact of RNA degradation on gene expression of 8-oxoguanine DNA glycosylase in human placenta from pregnancies complicated by preeclampsia and gestational diabetes mellitus and their gestation-matched controls. The data presented here show how RNA quality and appropriate reference gene selection is not only important to obtain accurate and reproducible RT-qPCR data but how different and even opposite results can be reported if the key steps outlined in the MIQE guidelines are not followed. The procedures and associated results presented in this study provide the first practical application of the MIQE guidelines to placental analysis in normal and pathological pregnancies.
authors.affiliation: INRS-Institut Armand-Frappier, Université du Québec, 531, boul. des Prairies, Building 18, Laval, QC, H7V 1B7, Canada.
authors.forename: Dave
authors.initials: D
authors.lastname: Lanoix
authors.forename: Andrée-Anne
authors.initials: AA
authors.lastname: Lacasse
authors.forename: Joey
authors.initials: J
authors.lastname: St-Pierre
authors.forename: Sean C
authors.initials: SC
authors.lastname: Taylor
authors.forename: Maude
authors.initials: M
authors.lastname: Ethier-Chiasson
authors.forename: Julie
authors.initials: J
authors.lastname: Lafond
authors.forename: Cathy
authors.initials: C
authors.lastname: Vaillancourt
mesh_terms: D000328:Adult; D045647:DNA Glycosylases / Q000235:genetics / Q000378:metabolism*; D016640:Diabetes, Gestational / Q000235:genetics; D005260:Female; D015870:Gene Expression; D020869:Gene Expression Profiling; D017408:Guidelines as Topic; D006801:Humans; D008297:Male; D010920:Placenta / Q000378:metabolism*; D011225:Pre-Eclampsia / Q000235:genetics; D011247:Pregnancy; D011786:Quality Control; D012313:RNA / Q000302:isolation & purification; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods / Q000592:standards*; D012015:Reference Standards; D012107:Research Design / Q000592:standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000592:standards*; D017422:Sequence Analysis, DNA; D013048:Specimen Handling; D055815:Young Adult
chemical_list: D012313:RNA; D045647:DNA Glycosylases; C479058:oxoguanine glycosylase 1, human