Development of COL3A1-Specific Real-Time PCR Assay for Clinical and Sport Detection Applications

Abstract

PIIINP, whose gene is COL3A1, is a protein used as a biomarker, which is altered in various pathologies such as fatty liver of non-alcoholic origin and is also used to detect abuse of human recombinant growth hormone (rhGH) by elite athletes to improve their performance. Therefore, the study of the levels of this protein or its gene could be useful for the diagnosis of these pathologies, in addition to other uses. The objective of this work was the development of a new detection method of the COL3A1 gene based on the quantification of mRNA in real time. 10 healthy men volunteers were injected intravenously for 3 consecutive days with 0.026 mg-1kg-1day-1person-1 of rhGH, and serum and blood samples were collected for 7 days at different times. mRNA extraction was performed after lymphocyte purification following standard protocols, and samples were quantified by quantitative real-time PCR using TaqMan probes designed specifically for this gene. The results showed an increase in COL3A1 expression that was maintained during 7 days of sampling after the last administration, observing more sensitive and robust results when compared to quantification of the PIIINP protein in serum from the same individuals. After the trial set-up, a partial validation was carried out in which parameters such as linearity, inter and intra-assay variation, influence of sex and age, stability of blood samples storage at 4°C and amount of blood sample, were studied.