Humanht 12 v3 beadchip

RNA abundance was measured in LCLs of 778 female individuals from the TwinsUK cohort using the Illumina Human HT-12 V3 BeadChip as part of the MuTHER project as previously described [49 (link)]. We selected 30 probes from GGT and PLA2 genes. Probes were adjusted for batch effects by linear models and residuals were inverse normalized prior to analysis.

RNA abundance was measured in abdominal fat samples using the Illumina Human HT-12 V3 Bead chip as part of the MuTHER project as previously described55 (link). The probe intensities were adjusted for batch effects using linear models prior to analysis. A previous study found 188 genes (199 probes) significantly associated with chronological56 (link) age. We performed stepwise regression to select expression probes independently associated with age. This procedure left 24 probes from 24 different genes (see Supplementary Table S1 for full list) for further analysis.

RNA was prepared from whole blood collected and stored in PAXgene Blood RNA Tubes (BD, Heidelberg, Germany) using the PAXgene Blood miRNA Kit (Qiagen, Hilden, Germany). Isolation of RNA was performed using a QIAcube according to protocols provided by the manufacturer Qiagen. Purity and concentration of RNA were determined using a NanoDrop ND-1000 UV-Vis Spectrophotometer (Thermo Scientific, Hennigsdorf, Germany). To ensure a consistently high RNA quality, all preparations were analyzed using RNA 6000 Nano LabChips on a 2100 Bioanalyzer (both from Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer's instructions. Samples exhibiting a RNA integrity number (RIN) less than seven were excluded from subsequent analyses. The Illumina TotalPrep-96 RNA Amplification Kit (Ambion, Darmstadt, Germany) was used for reverse transcription of 500 ng RNA into double-stranded (ds) cDNA and subsequent synthesis of biotin-UTP-labeled antisense-cRNA using this cDNA as the template. Finally, in total 3,000 ng of cRNA were hybridized with a single array on the Illumina HumanHT-12 v3 BeadChips, followed by washing and detection steps in accordance with the Illumina protocol. BeadChips were scanned using the Illumina Bead Array Reader. Further details on expression data transformation and quality control are available elsewhere [11 (link)].