Context The endocrine function of human fetal adrenals (HFAs) is activated already during first trimester, but adrenal steroidogenesis during fetal life is not well characterized. of 3 minutes at 95C, 25 cycles of 30 seconds at 95C, 1 minute at 60C, 1.5 minutes at 65C, and one cycle of 5 minutes at 72C. Table 1. Primers Utilized for RT-PCR and CA-074 Methyl Ester novel inhibtior Quantitative PCR aldo-keto reductase family 1 member C3; HGNC, HUGO Gene Nomenclature Committee. Gene expression Total RNA was extracted from one frozen HFA gland in samples from GWs 8 to 12 (11 male and 11 female, collected from 22 fetuses), whereas half of a frozen HFA gland was used in samples from GWs 14 to 19 (9 male and 8 female, collected from 17 fetuses) and isolated using the NucleoSpin RNA II purification kit according to the manufacturers instructions (Macherey-Nagel, Dren, Germany). cDNA was synthesized using a dT20 primer and random hexamers. Real-time polymerase chain reaction (RT-PCR) was performed using specific primers targeting preselected mRNAs. All primers were designed CA-074 Methyl Ester novel inhibtior to span intron-exon boundaries with optimal annealing temperatures of 62C, comparable primer length, and CG contents (Table 1). All amplicons were initially verified by sequencing (Eurofins MWG GmbH, Ebersberg, Germany), and primer amplification efficiency and CA-074 Methyl Ester novel inhibtior detectable dynamic range of all primer units were validated before the analysis of the HFA samples. RT-PCR cycle conditions were as follows: one cycle of 3 minutes at 95C, 40 cycles of 30 seconds at 95C, 1 minute at 62C, 1 minute at 72C, and one cycle of 5 minutes at 72C. Quantitative RT-PCR analysis was performed in triplicate using Amazing CA-074 Methyl Ester novel inhibtior II SYBR Green qPCR Grasp Mix (Agilent Technologies, Santa Clara, CA). Changes in gene expression were quantified using the 2 2?(4C). Each tube was transferred to a dry ice bath (dry ice pills in ethanol, 99%) for a few minutes to freeze the aqueous phase, followed by decantation of the organic phase to a new glass tube. The organic phase was evaporated to dryness under a stream of N2, and finally, the steroids were resolved in an appropriate amount of 50% (v/v) MeOH (tissue GWs 8 to 12: 100 L; tissue GWs 14 to 19: 200 L) for LC-MS/MS analysis as previously explained (22). All samples were measured in one single batch, which included requirements for calibration curves, unknown samples, and two blanks and for method control; three unspiked human serum pool samples and three serum pool samples spiked with low and high levels, respectively. Statistical analysis Quantitative PCR and LC-MS/MS data were statistically analyzed for age- and sex-specific differences. Age differences were tested by the nonparametric Mann-Whitney test in which the HFA age groups GWs 10 to 12, GWs 14 to 16, and GWs 17 to CA-074 Methyl Ester novel inhibtior 19 were compared with male GWs 8 to 9. Sex differences were also tested by the nonparametric Mann-Whitney test within each age group. 0.05 was considered statistically significant. Results Gene expression patterns of adrenal steroidogenic enzymes The selected steroidogenic enzymes were expressed in all investigated HFA glands at the transcriptional level. Gene expression patterns were investigated separately in male Spp1 and female samples, which were divided into four age groups: GWs 8 to 9, GWs 10 to 12, GWs 14 to 16, and GWs 17 to 19. Expression levels were calculated as a ratio of levels relative to male GWs 8 to 9 (reference value set to 1 1). No sex differences were observed in the transcription levels of the examined steroidogenic enzymes or in the transcription levels of the ACTH receptor, and increased approximately 10-fold, whereas the expression level of and increased even further, by 50-fold and 20-fold, respectively (Fig. 1e and 1f). Only two of the investigated steroidogenic enzymes were constitutively expressed throughout the first and second trimesters, namely and aldo-keto reductase family 1 member C3 (also known as 17and were lower than those of the remaining steroidogenic enzymes (Fig. 1j). Open in a separate window Physique 1. Gene expression level of human being fetal adrenal steroidogenic enzymes through the second and 1st trimesters. (a?we) Quantitative change transcription polymerase string reaction evaluation of a variety of steroidogenic-associated enzymes and receptors in man and female human being fetal adrenal samples split into four age ranges: GWs 8 to 9, GWs 10 to 12, GWs 14 to 16, and GWs 17 to 19. Manifestation is in accordance with the research gene 0.05; ** 0.01. (j) General human being fetal adrenal transcript.