Transcripts, proteins and metabolites in rat adipose tissue

[ENG:]The dataset is for Wistar rats and includes measurements taken on postnatal days (PND) 30, 35, 40, 46, and 60. It contains information on the expression of genes associated with adipose tissue browning (Cidea, Ucp1, Prdm16), levels of cholinergic pathway proteins (CHAT, CHRNA2, UCP1), as well as free and total choline and acetylcholine concentrations, and liver choline content. The data are arranged in a single table. Rows contain case names, and columns contain variable names and measured parameters. Methods: Choline and acetylcholine concentrations in inguinal adipose tissue were determined using the Choline/Acetylcholine Quantification Kit, Sigma Aldrich, cat. no. MAK056.Hepatic choline concentration was determined using the Choline Assay Kit, cat. no. MAK508, Sigma Aldrich.Real-time PCRTotal RNA was isolated from inguinal adipose tissue and liver using the commercial Extrazol kit (Blirt) according to the manufacturer's protocol. RNA quality and quantity were assessed using a microvolume spectrophotometer (DS-11, DeNovix, USA).cDNA synthesis was performed using 2 μg of RNA using the Transcriptor First Strand Synthesis Kit (Roche, Germany). Real-time reactions were performed in duplicate on a LightCycler 480 (Roche) using TaqMan™ Gene Expression Assay probes (Thermo Fisher Scientific, USA) and Fast Advanced Master Mix for qPCR (Thermo Fisher Scientific, USA).Gene transcription levels were analyzed using the following probes: Cidea (Rn04181355_m1, Thermo Fisher Scientific, USA), Ucp1 (Rn00562126_m1, Thermo Fisher Scientific, USA), Prdm16 (Rn01516224_m1, Thermo Fisher Scientific, USA). Two reference genes were used for data normalization. The arithmetic mean of the transcript levels of two reference genes was used for normalization: Tbp (Rn01455646_m1, Thermo Fisher Scientific, USA) and Actb (Rn00667869_m1, Thermo Fisher Scientific, USA).Relative quantitative analysis of mRNA levels was performed using the maximum second derivative method.Protein IsolationProteins were isolated from 100 mg of tissue. Mechanical homogenization was performed in ice-cold PBS. Samples were centrifuged at 10,000 g for 5 minutes. Protein concentration was determined using a bicinchoninic acid (BCA) kit (Thermo Scientific).Protein Concentrations in Adipose TissueThe concentrations of UCP1, CHAT, and CHRNA2 proteins were determined using kits: Rat UCP1 (Uncoupling Protein 1, Mitochondrial) ELISA Kit, cat. no.: 6326, ELK Biotechnology; Rat CHRNA2 (Neuronal Acetylcholine Receptor Subunit Alpha-2) ELISA Kit, cat. no.: ELK0108, ELK Biotechnology; Rat CHAT (Choline Acetyltransferase) ELISA Kit, cat. no.: ELK5756, ELK Biotechnology. Analyses were performed according to the manufacturer's protocol.