Extraction of RNA and quantitative real-time PCR in scAT and liver samples

Details of the RNA extraction and cDNA synthesis were described previously⁵¹. After tissue homogenization with the Precellys 24 system (VWR/Peqlab Biotechnologie, Erlangen, Germany) total RNA was extracted from liver and scAT by using the TRIzol reagent (Invitrogen/Life Technologies, Carlsbad, CA, USA) according to the manufacturer´s protocol. The RNA was purified with spin columns according to the Qiagen kit protocol (RNeasy Mini Kit, Qiagen GmbH, Hilden, Germany). The concentration of total RNA and the purity was quantified at 260 nm and 280 nm using the Nanodrop 1000 (peQLab Biotechnology GmbH, Erlangen, Germany). For cDNA synthesis, a reverse transcription of 250 ng total RNA per 20 µL reaction volume was performed with RevertAid reverse transcriptase (Thermo Scientific GmbH, Dreieich, Germany) according to the manufacturer’s instructions with a Multicycler PTC 200 (MJ Research Inc, Watertown, MA, USA). Quantitative real-time PCR (qPCR) was carried out using an MX3000p PCR cycler (Stratagene, Amsterdam, the Netherlands, and Agilent, Santa Clara, CA, USA) in accordance with the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines⁵⁵. The qPCR conditions and primer sequences used in the present study are presented in Table ​Table1.1. Samples were run as triplicates in a total volume of 10 µL, with 2 µL of cDNA (diluted 1:4) as a template, 1 µL of assay-specific primer mix, 2 µL of water, and 5 µL of DyNAmo ColorFlash SYBR Green qPCR Kit (Thermo Fisher Scientific, Dreieich, Germany). Each run included a negative-template control for quantitative PCR, as well as a negative-template control and no-reverse-transcriptase control of cDNA. Relative quantification of the target genes, i.e. HSD11B1, HSD3B1, HSD17B12, StAR, and CYP21 was performed with standard curves using cDNA serial dilutions to calculate the abundance based on run-specific PCR efficiency. For each PCR, a set of two inter-run calibrators was used to correct for inter-run variation.

Primer characteristics of target and reference genes used in adipose tissue and liver and the real-time polymerase chain reaction conditions.

¹HSD17B12 = 17 ß-hydroxysteroid dehydrogenase type 12; CYP21 = steroid 21-hydroxylase; StAR = steroidogenic acute regulatory protein; HSD3B1 = 3β-Hydroxysteroid dehydrogenase type 1; HSD11B1 = 11ß-hydroxysteroid dehydrogenase type 1.

²LRP10 = lipoprotein receptor-related protein 10; POL2 = RNA polymerase II; HPCAL1 = hippocalcin-like protein 1; EIF3K = eukaryotic translation initiation factor 3 subunit K; EMD = Emerin.

³Base pairs.

⁴Initial denaturation for 10 min at 90 °C; denaturation for 30 s at 95 °C; 40 cycles, except for StAR, HSD3B1, and HPCAL1 (35 cycles), LRP10 (33 cycles).

⁵Extension at 72 °C.

Target genes were normalized based on the most stable reference genes in liver and scAT, i.e. low-density lipoprotein receptor-related protein 10 (LRP10), RNA polymerase II (POL2), eukaryotic translation initiation factor 3 subunit K (EIF3K), hippocalcin-like protein 1 (HPCAL1), and emerin (EMD), determined by geNorm^PLUS algorithms of qBASE^plus 3.1 software (Biogazelle, Ghent, Belgium).