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.

Urinary Exosomal MicroRNA Signatures in Nephrotic, Biopsy-Proven Diabetic Nephropathy

“…A major limitation of our study was the lack of real-time quantitative PCR-based validation of our identified miRs. However, unlike the known-reference miRs in cervical cancer and colorectal cancer [41,42], the reference miRs in DN remain unknown. An alternative approach to overcome this problem we would like to suggest is to test our reported deregulated miRs in various clinical and experimental models.…”

Reduced Expression of IL-1β and IL-18 Proinflammatory Interleukins Increases the Risk of Developing Cervical Cancer

“…Total RNA was purified in a subsequent stage by means of the miRNA Absolutely RNA Kit (Agilent Technologies). The RNA quality was assessed by a NanoDrop 2000 Spectrophotometer (ThermoScientific, Wilmington, DE, USA) and 1% agarose gel electrophoresis (Bustin et al, 2009;Leitão et al, 2014;Rueda-Martínez et al, 2014). After this, 1 μg of RNA samples of a suitable quality (an OD260/280 from 1.8 to 2.1 and intact rRNA subunits -28S and 18S) was used to generate the cDNA with the aid of a miScript II RT kit (Qiagen).…”

“…Reference genes that had been previously validated by the group of cervical tissues, were used to obtain mRNA expression levels (26???). In this way, the geometric mean of GAPDH and the ACTB reference genes, was used to calculate the relative expression of IL-1β and IL-18 mRNAs (Leitão et al, 2014). Every qPCR reaction was run in duplicate for each sample to minimize pipetting error (Nolan et al, 2006).…”

Immune Regulatory Network in Cervical Cancer Development: The Expanding Role of Innate Immunity Mechanisms

“…At the mRNA level, STING expression was analyzed in ficoll-isolated PBMC using semi-qPCR (RPLP0 and PGK1 genes were used as endogenous controls [26]): similar to flow cytometry results, in PBMC from patients with preinvasive/microinvasive cancer (stage 0-IA), STING-mRNA Figure 7A) suggesting the need for T cell (CD4/CD8) separation in further analysis. STING-mRNA expression was also assessed in samples of HPV-negative morphologically normal epithelium (control), HPV-positive precancerous lesions of the cervix, carcinoma in situ and microinvasive carcinoma (relative to four genes-EEF1A1, ACTB, GAPDH, and RPLP0-taken as endogenous controls due to their proved constitutive expression in cervical tissues [27]) ( Figure 7B). In contrast to lymphocytes, a considerable (up to 50%) proportion of pathological samples in each patient group showed elevated STING expression as compared with normal noninfected epithelium (though the group mean values did not differ statistically).…”