Quantification of hormone membrane receptor FSHR, GPER and LHCGR transcripts in human primary granulosa lutein cells by real-time quantitative PCR and digital droplet PCR

Abstract

BackgroundQuantitative real time polymerase chain reaction (qPCR) and droplet digital PCR (ddPCR) are methods used for gene expression analysis in several contexts, including reproductive endocrinology. ObjectivesHerein, we compared qPCR and ddPCR technologies for gene expression analysis of hormone membrane receptor-encoding genes (FSHR, GPER and LHCGR), as well as the commonly used RPS7 housekeeping gene, in order to identify the most reliable method to be applied for gene expression analysis in the context of human reproduction. MethodsTotal RNA was extracted from human primary granulosa lutein cells of donor patients undergoing assisted reproduction and used for gene expression analysis by qPCR and ddPCR, after finding the optimal annealing temperature. Immunostaining for protein localization in cell membranes was also performed. ResultsBoth techniques provided results reflecting the low number of FSHR and GPER transcripts, although ddPCR quantified the low-expressed genes with major accuracy, thanks to its higher reaction efficiency. The absolute FSHR and GPER transcript number was also determined by ddPCR, resulting in 40- to 260-fold lower amount than LHCGR transcripts. qPCR and ddPCR data are convergent with immunofluorescence analysis of membrane receptor expression in human primary granulosa lutein cells. ConclusionThese results suggest that ddPCR is the candidate technology for analysis of genes with relatively low expression levels and provides useful insights for characterizing hormone receptor expression levels in the context of reproductive endocrinology.