Sequence detection system 2

The total RNA was extracted from the hypothalamus, liver, spleen, isolated bone marrow cells, intraperitoneal macrophages, and cell line BV-2 using Trizol® Reagent (Invitrogen Corporation, CA, USA) according to the manufacturer's recommendations and quantitated using a ND-2000 Nanodrop (Thermo Electron, WI, USA). Reverse transcription was performed with 3 μg of the total RNA, using a High Capacity cDNA Reverse Transcription kit (Life Technologies Corporation, Carlsbad, CA, USA). The relative expression was determined using the TaqMan™ detection system, and the primers for the target genes were obtained from Applied Biosystems: Mm01312230_m1 for CHRNA7; Mm00446190_m1 for IL-6; Mm00434228_m1 for IL-1β; and Mm00443258_m1 for TNFα. GAPDH (4351309) or β-actin (4351315) was used as endogenous controls. Gene expression was quantitated by performing real-time PCR on an ABI Prism 7500 Fast platform. The data were analyzed using a Sequence Detection System 2.0.5 (Life Technologies Corporation, Carlsbad, CA, USA) and expressed as relative values determined by the comparative threshold cycle (Ct) method (2–ΔΔ Ct), according to the manufacturer's recommendations.

Total epididymal white adipose tissue (WAT) RNA was extracted using TRIzol reagent (Life Technologies Corporation, CA, USA) according to the manufacturer’s recommendations and was quantified using a NanoDrop ND-2000 (Thermo Electron, WI, USA). Reverse transcription was performed with 3 ng of total RNA, using a high-capacity cDNA reverse transcription kit (Life Technologies Corporation, CA, USA). Relative expression levels were determined using a Taqman detection system and primers for the target genes (Table 3).
Each PCR reaction contained 20 ng of complementary DNA. Real-time PCR was performed on an ABI Prism 7500 Fast platform. Data were analysed using the sequence detection system 2.0.5 (Life Technologies Corporation, CA, USA) and expressed as relative values determined by the comparative threshold cycle (Ct) method (2−ΔΔCt), according to the manufacturer’s recommendations.

Total RNA was extracted from the white gastrocnemius and soleus muscles using Trizol reagent (Life Technologies Corporation, CA, USA) according to the manufacturer's recommendations. Total RNA was quantified using a Nanodrop ND-2000 (Thermo Scientific, WI, USA). Reverse transcription was performed with 3 μg of total RNA using a High-Capacity cDNA Reverse Transcription kit (Life Technologies Corporation, California, USA). The messenger RNA (mRNA) was measured using primers and a TaqMan detection system obtained from Applied Biosystems: MCT-1 (Slc16a1, GenBank accession NM_012716) and MCT-4 (Slc16a3, GenBank accession NM_030834). Glyceraldehyde-3-phosphate dehydrogenase primer (Applied Biosystems, cat n# 4352338E) was used as an endogenous control. Real-time PCR analysis of the gene expression was performed on an ABI Prism 7500 Fast platform using 20 ng of cDNA. The data were analyzed using a Sequence Detection System 2.0.5 (Life Technologies Corporation, CA, USA) using the comparative threshold cycle method (2^−ΔΔCt), according to the manufacturer's recommendation.

The total RNA was extracted from the hypothalamus, liver, and spleen using Trizol^® Reagent (Invitrogen Corporation, CA, USA) according to the manufacturer's recommendations, and quantitated using a Nanodrop ND-2000 (Thermo Electron, WI, USA). Reverse transcription was performed with 3 μg total RNA and a High Capacity cDNA Reverse Transcription kit (Life Technologies Corporation, Carlsbad, CA, USA). Relative expression was determined using the TaqMan™ detection system and primers for the target genes: Mm01312230_m1 for CHRNA7; Mm00443258_m1 for TNF-α; Mm00434228_m1 for IL-1β; Mm00446190_m1 for IL-6; Mm01288386_m1 for IL-10; Mm00657889_mH for Chil3; Mm00436450_m1 for CXCL2; Mm00441242_m1 for CCL2; and Mm00436454_m1 for CX3CL1 (Life Technologies Corporation, Carlsbad, CA, USA). GAPDH was used as the endogenous control (4352339E mouse GAPDH, Life Technologies Corporation, Carlsbad, CA, USA). Each PCR reaction contained 20 ng cDNA. Gene expression was quantitated by real-time PCR performed on an ABI Prism 7500 Fast platform. Data were analyzed using a Sequence Detection System 2.0.5 (Life Technologies Corporation, Carlsbad, CA, USA), and expressed as relative values determined by the comparative threshold cycle (Ct) method (2–ΔΔ Ct), according to the manufacturer's recommendations (16 (link)).

RNA from P19 cortices from anx/anx, anx/+ and +/+ mice was isolated using Trizol reagent (Invitrogen) according to manufacturer's instructions. cDNA was synthesized from total RNA using oligo-dT primers and the Superscript III First Strand Synthesis System (Invitrogen) according to manufacturer's instructions (2 µg of RNA used in 20 µl reverse-transcription reaction). Primers specific for Tyro3 (5′-AATGCCGAGATTTACAACTACCTCAT-3′ and 5′-GTTCCATTCGACAGACACGTGAAGCTT-3′) and GAPDH (5′-TGTGAACGGATTTGGCCGTAT-3′and 5′-CATGTAGACCATGTAGTTGAG-3′) were used. Reactions were run with SYBR green PCR master mix (Invitrogen) in an AB 7500 fast real-time PCR system and relative gene expression levels were calculated using Sequence Detection System 2.2 Software (Applied Biosystems). Expression values were normalized relative to sample Gapdh mRNA expression. RNA from five anx/+ and +/+ and three anx/anx animals was analyzed. Data were analyzed using nonparametric t-tests and Mann–Whitney test.

All 138 patients were genotyped for four functional polymorphisms of the GR gene (ER22/23EK, rs6189 and rs6190; N363S, rs6195; BclI, rs41423247 and 9β, rs6198) and the GLCCI1 gene variant (rs37972). DNA was extracted from samples of peripheral venous blood samples using standard techniques. Genotyping was performed using Taqman allelic discrimination assays (Applied Biosystems), following protocols described by the supplier. Genotyping of BclI failed in one patient, in two patients each the N363S, and GLCCI1 genotype could not be determined. Results were analyzed using the sequence detection system 2.2 software (Applied Biosystems).