Establishment of standard curves useful for quantitation of the expression of nuclear receptors mRNA

• Grant number: 13/14176-3
• Support Opportunities: Regular Research Grants
• Duration: March 01, 2014 - February 29, 2016
• Field of knowledge: Health Sciences - Medicine
• Principal Investigator: Flavio Richeti
• Grantee: Flavio Richeti
• Host Institution: Faculdade de Ciências Médicas da Santa Casa de São Paulo (FCMSCSP). Fundação Arnaldo Vieira de Carvalho. São Paulo , SP, Brazil
• Associated researchers: Carlos Alberto Longui ; Elisangela Oliveira da Silva ; Mylene Neves Rocha

Abstract

Several methods have been developed in order to quantify genes expression. Fragment amplification techniques such as real-time polymerase chain reaction (RT-PCR) are employed, allowing the quantification of messenger RNA (mRNA) and recognition of its tissue-specific expression. The mRNA quantification by RT-PCR requires stringent criteria to ensure the reproducibility of results. One such criteria is a standard curve derived from cells expressing a significant and stable amount of sequence of interest, resulting in absolute quantification of gene expression. Regarding to nuclear receptors, there are limitations in the selection of a single cell type expressing at the same time suitable mRNA of androgen receptor, glucocorticoid receptor and estrogen receptor. This requires a different cellular origin for each receptor type, which introduces an undesirable variation among standard curves, reducing the reproducibility of results over time and in different laboratories. In this study, the aim is to amplify sufficient amounts of cDNA containing the sequence of interest, of each gene subsequently stored in aliquots capable to be used as reference standard curves for analysis of androgen receptor genes, glucocorticoid receptor genes (alpha and beta isoforms), estrogenic receptor and BCR (Breakpoint Cluster Region) normalizing gene. (AU)