Taqman human gene expression assays

Selected microarray results were confirmed via real-time polymerase chain reaction (qPCR) in a larger cohort of 34 (Ob = 19, L = 15; Additional file 1: Table S1) subjects with available RNA. No statistical differences in demographics were found between this larger cohort and either microarray cohort. RNA (2μg) was reversed-transcribed into cDNA using the SuperScript III Reverse Transcription kit (Invitrogen Corp.; Carlsbad, CA). PCR was performed in triplicate on an Applied Biosystems 7900HT Fast Real-Time PCR System with Taqman Universal PCR Master Mix and commercially available TaqMan human gene expression assays (ThermoFischer Scientific; Waltham, MA) for protein phosphatase 2 regulatory subunit B gamma (PPP2R5C; AssayID:Hs00604899_g1) and transcription factor A, mitochondrial (TFAM; AssayID:Hs00273372_s1). Assays were performed in accordance with manufacturer instructions: 50 °C for 2 min, 95° for 10 min, followed by 40 cycles of 95 °C for 15 s followed by 60 °C for 1 min. mRNA content was determined via the comparative C_t methodology. Fold changes between Ob and L groups were determined via the 2^−ΔΔCt methodology where ΔΔC_t = ΔC_{t Obese}−ΔC_{t Lean}. Assays were run with a multiplexed endogenous control (18S RNA).

Microarray results were confirmed with real-time polymerase chain reaction (qPCR). Due to RNA quantity and concentrations available following microarray analysis, a representative subset of n = 3 in the T2D group was used in qPCR analysis. RNA (100 ng) was reverse-transcribed into cDNA using SuperScript III Reverse Transcription (Invitrogen Corp.; Carlsbad, CA) following manufacturer protocols. PCR was performed in triplicate on an Applied Biosystems QuantStudio 3 Real-Time PCR Systems with Taqman Universal PCR Master Mix and commercially available TaqMan human gene expression assays (ThermoFisher Scientific; Waltham, MA) for hexokinase 2 (HK2; AssayID: Hs00606086_m1), NADH:ubiquinone oxidoreductase subunit B8 (NDUFB8; Hs00428204_m1), NADH:ubiquinone oxidoreductase subunit B7 (NDUFB7; Hs00958815_g1), NADH:ubiquinone oxidoreductase subunit A1 (NDUFA1; Hs00244980_m1), and 3-hydroxybutyrate dehydrogenase, type 1 (BDH1; Hs00366297_m1). Assays were performed in accordance with manufacturer instructions: 50°C for 2 min, 95° for 10 min, followed by 40 cycles of 95°C for 15 s followed by 60°C for 1 min. Assays were run with a multiplexed endogenous control (B2M). Fold changes were determined via the 2^−ΔΔCt methodology.