title: Quantifying mRNA and microRNA with qPCR in cervical carcinogenesis: a validation of reference genes to ensure accurate data.
abstract: A number of recent studies have catalogued global gene expression patterns in a panel of normal, tumoral cervical tissues so that potential biomarkers can be identified. The qPCR has been one of the most widely used technologies for detecting these potential biomarkers. However, few studies have investigated a correct strategy for the normalization of data in qPCR assays for cervical tissues. The aim of this study was to validate reference genes in cervical tissues to ensure accurate quantification of mRNA and miRNA levels in cervical carcinogenesis. For this purpose, some issues for obtaining reliable qPCR data were evaluated such as the following: geNorm analysis with a set of samples which meet all of the cervical tissue conditions (Normal + CIN1 + CIN2 + CIN3 + Cancer); the use of individual Ct values versus pooled Ct values; and the use of a single (or multiple) reference genes to quantify mRNA and miRNA expression levels. Two different data sets were put on the geNorm to assess the expression stability of the candidate reference genes: the first dataset comprised the quantities of the individual Ct values; and the second dataset comprised the quantities of the pooled Ct values. Moreover, in this study, all the candidate reference genes were analyzed as a single "normalizer". The normalization strategies were assessed by measuring p16INK4a and miR-203 transcripts in qPCR assays. We found that the use of pooled Ct values, can lead to a misinterpretation of the results, which suggests that the maintenance of inter-individual variability is a key factor in ensuring the reliability of the qPCR data. In addition, it should be stressed that a proper validation of the suitability of the reference genes is required for each experimental setting, since the indiscriminate use of a reference gene can also lead to discrepant results.
authors.affiliation: Laboratory of Molecular Studies and Experimental Therapy (LEMTE), Department of Genetics, Center for Biological Sciences, Federal University of Pernambuco, Pernambuco, Brazil.
authors.forename: Maria da Conceição Gomes
authors.initials: Mda C
authors.lastname: Leitão
authors.affiliation: Laboratory of Molecular Studies and Experimental Therapy (LEMTE), Department of Genetics, Center for Biological Sciences, Federal University of Pernambuco, Pernambuco, Brazil.
authors.forename: Eliane Campos
authors.initials: EC
authors.lastname: Coimbra
authors.affiliation: Laboratory of Molecular Studies and Experimental Therapy (LEMTE), Department of Genetics, Center for Biological Sciences, Federal University of Pernambuco, Pernambuco, Brazil.
authors.forename: Rita de Cássia Pereira
authors.initials: Rde C
authors.lastname: de Lima
authors.affiliation: Clinical Hospital of Federal University of Pernambuco, Pernambuco, Brazil.
authors.forename: Mariléa de Lima
authors.initials: Mde L
authors.lastname: Guimarães
authors.affiliation: Institute of Integral Medicine Prof. Fernando Figueira, Pernambuco, Brazil.
authors.forename: Sandra de Andrade
authors.initials: Sde A
authors.lastname: Heráclio
authors.affiliation: Molecular and Cytological Research Laboratory, Department of Histology, Federal University of Pernambuco, Pernambuco, Brazil.
authors.forename: Jacinto da Costa
authors.initials: Jda C
authors.lastname: Silva Neto
authors.affiliation: Laboratory of Molecular Studies and Experimental Therapy (LEMTE), Department of Genetics, Center for Biological Sciences, Federal University of Pernambuco, Pernambuco, Brazil.
authors.forename: Antonio Carlos
authors.initials: AC
authors.lastname: de Freitas
mesh_terms: D002471:Cell Transformation, Neoplastic / Q000235:genetics*; D002584:Cervix Uteri / Q000378:metabolism; D005260:Female; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D035683:MicroRNAs / Q000235:genetics*; D020871:RNA Stability; D012333:RNA, Messenger / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results; D002583:Uterine Cervical Neoplasms / Q000235:genetics*
chemical_list: D035683:MicroRNAs; D012333:RNA, Messenger