Can the brain be taken as a whole when choosing housekeeping genes for studding sex differences?

DADAM F; VIVAS LAURA; CAEIRO XIMENA

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Congreso; Reunión Anual 2121-SAFIS; 2021

SAFIS

Can the brain be taken as a whole when choosing housekeeping genes for studying sex differences?Dadam F.1,3, Vivas L.1,2, Caeiro XE11Instituto de Investigación Médica Mercedes y Martin Ferreyra-INIMEC-CONICET-UNC2Facultad de Ciencias Exactas Físicas y Naturales- UNC3Facultad de Psicología - UNCExpression of sexual dimorphic genes in differente brain areas are been extensively studied. But can the brain be taken as a whole when chossing housekeeping genes (HKG) for studing sex differences? This study aimed to define whether sex chromosome complement (SCC) and/or the organizational hormonal effects may differentially modulate relative gene expression of three common used HKG, glyceraldehyde-3-phosphate dehydrogenase (Gapdh), Ciclophylin (Cyc) and ribosomal subunit 18S (18s) in different brain areas.For this purpose, we used mice of the "four core genotypes" mouse model, in which the effect of gonadal sex (testes/ovaries) and SCC (XX/XY) are uncoupled. Brains were collected from gonadectomized mice and relative gene expression of basal Gapdh, Cyc and 18s genes at fore/hindbrain nuclei were assessed using quantitative real time-PCR. Data were subjected to a 2-way mixed ANOVA with gonadal sex (male/female) and SCC (XY/XX) as independent factors. Furthermore, to identify the HKG with the most stable expression; HKGs stability test- geNorm and Normfinder- were used.Sex-dependent expressions of commonly used housekeeping genes at the SFO and AP brain levels were observed. At SFO Gapdh and Cyc genes showed a female phenotype. Furthermore, statistical data demonstrated a SCC effect on Cyc gene expression in the SFO as well as for 18s at the AP. At the NTS, OVLT and PVN no differences in gene expression were observed. Besides, the analysis of the M-value for each HKG demonstrated not only the importance of taking into account sex as a factor (since the stability values were always much higher when males and females worked together) but also studying each area independently (obtaining in this way lower M-values than working all the brain tissue together). Our findings underscore the importance of empirical determination of reference genes to effectively and accurately normalize RT-PCR data before quantifying target genes.