PO-393 Identification of housekeeping genes to quantitative real-time RT-PCR analysis by miRNA expression using liquid-based cervical cytologysamples

Abstract

IntroductionSome studies have showing that cervical cytology is limited as a single method for the screening of cervical cancer. In view of this, it would be extremely important to incorporate molecular tests that could improve accuracy for detection of precursor lesions. In this scenario the evaluation of the differential expression of microRNAs (miRNAs) open a window of opportunities to understand tumour onset and progression considering that can be used are good noninvasive biomarkers detected in liquid biopsy samples, such as liquid-based cervical cytology (LBC) samples. However, a few studies have been carried out in these kind of samples to detect the expression profile of miRNAs in cervical cancer precursor lesions. Furthermore, the most of the studies used a small samples or evaluated a few groups of miRNAs. The analysis of miRNAs profile would allow to open diagnostic testing arsenal for the screening of Cervical Intraepithelial Neoplasia (CIN). In view of the above, there is a need for technical standardisation for the analysis of miRNA expression in LBC samples considering the poor evidence in the literature that used LBC samples for the analysis of miRNA expression. Nevertheless, the aimed of this study was to identify housekeeping’s for analysis of miRNA expression in LBC samples.Material and methodsExpression of U6, hsa-miR-16 RNU-44, 47, 48 and 49 was measured by Reverse Transcriptase Quantitative PCR (RT-qPCR). Reference genes expression values were normalised to the reference using the comparative CT method (2-CT). We used one common software, namely NormFinder, to analyse expression stability of the six selected genes. This software candidate reference genes by calculating their stability values, with lower values indicating more stable genes.Results and discussionsThe stability values calculated using NormFinder to six housekeepings was (U6=0,787; miR-16=9,728; RNU-44=7,912; RNU-47=3,234; RNU-48=5,132; RNU-49=1,716) and the standard error (U6=2,127; miR-16=1,990; RNU-44=1,684; RNU-47=1,084; RNU-48=1,265; RNU-49=1,226). Thereby, we can verify that U6 was the gene that demonstrated to be the best housekeepin, this because it presented smaller variation in the expression in relation to the other genes tested. Similar data were demonstrated in the literature. However, it was the first time that it was performed with liquid-based cervical cytology samples.ConclusionWe conclude that the small nucleolar RNA transcript U6 was the best reference gene.