Nanodrop 1000

Liver RNA was extracted using RNA Bee Total RNA Isolation Kit (Bio-Connect, Huissen, the Netherlands) and Ambion Total RNA isolation was used for RNA extraction of WAT (AM1912, Life Technologies, Bleiswijk, The Netherlands). RNA concentration was assessed spectrophotometrically using Nanodrop 1000 (Isogen Life Science, De Meern, the Netherlands) and RNA quality was determined by Lab-on-Chip analysis using 2100 Bioanalyzer (Agilent Technologies, Amstelveen, the Netherlands). cDNA was synthesized from total RNA using a High Capacity RNA-to-cDNA™ Kit (Life Technologies). Gene expression analyses were performed by RT-PCR on an Applied Biosystems 7500 Fast Real-time PCR system. TaqMan® Gene Expression Assays (Life Technologies) were used to detect the expression of the following genes: CD68 (Mm03047340_m1), CD11c (Itgax, Mm00498698_m1), Arginase-1 (Arg1, Mm00475988_m1), Mcp-1 (Ccl2, Mm00441242_m1), Tnf-α (Tnf, Mm00443258_m1). Glyceraldehyde 3-phosphate dehydrogenase (Gapdh; 4308313), hypoxanthine-guanine phosphoribosyltransferase (Hprt; Mm00446968_m1) and peptidylprolyl isomerase F (Ppif; Mm00506384_m1) were used as housekeeping genes. Changes in gene expression were calculated using the comparative Ct (ΔΔCt) method and expressed as fold-change relative to HFD control group.

Extraction of total RNA from cord blood was performed according to the manufacturer’s instructions using the miRNeasy mini kit (Qiagen, Venlo, Netherlands). Subsequently, DNase treatment (Qiagen, Venlo, Netherlands) was performed and RNA quantity and purity were determined by spectrophotometric means (Nanodrop 1000, Isogen, Life Science, Belgium). RNA integrity was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Amstelveen, Netherlands). Fluorescent cyanine-3-labeled cRNA was synthesized from 0.2 μg total RNA according to the Agilent one-color Quick-Amp labeling protocol (Agilent Technologies). After hybridization onto Agilent Whole Human Genome 8 × 60 K microarrays signal detection was performed using the Agilent DNA G2505C Microarray Scanner (Agilent Technologies). Preprocessing of raw data was carried out by Agilent Feature Extraction Software (Version 10.7.3.1, Agilent Technologies, Amstelveen, Netherlands) and in-house software within the R statistical computing environment.

The DNA samples were prepared from bacteria grown on Columbia Sheep Blood agar plates (Oxoid) with the QIAGEN DNeasy blood and tissue kit (Qiagen) according to the manufacturer's instructions for Gram‐positive bacteria. Both DNA purity and concentration were assessed using the Nanodrop 1000 (Isogen Life Science) before storage at −20°C.

Total RNA was extracted from liver tissue using RNA Bee Total RNA Isolation Kit (Bio-Connect, Huissen, the Netherlands). Spectrophotometric analysis of RNA concentration was performed using Nanodrop 1000 (Isogen Life Science, De Meern, the Netherlands) and quality of RNA was assessed using a 2100 Bioanalyzer (Agilent Technologies, Amstelveen, the Netherlands). cDNA was synthesised using a High Capacity RNA-to-cDNA™ Kit (Life Technologies, Bleiswijk, the Netherlands). Hepatic gene expression analyses were performed by RT-PCR on a 7500 Fast Real-Time PCR System (Applied Biosystems by Life Technologies) using TaqMan® Gene Expression Assays (Life Technologies). Transcripts were quantified using TaqMan® Gene Expression Assays (Life Technologies) and the following primer/probe-sets for Srebf1 (Mm00550338_m1), Fasn (Mm00662319_m1), Dgat1 (Mm00515643_m1), Ppara (Mm00440939_m1), Cpt1a (Mm01231183_m1), Acox1 (Mm00443579_m1), Ccl2 (Mm00441242_m1), Tnf (Mm00443258_m1), Il1b (Mm00434228_m1) and the endogenous controls Hprt (Mm00446968_m1) and Ppif (Mm01273726_m1). Changes in gene expression were calculated using the comparative Ct (ΔΔCt) method and expressed as fold-change relative to CON.

Placental DNA was extracted from one placental tissue biopsy using the QIAamp DNA Mini Kit (Qiagen, Venlo, The Netherlands). We assessed DNA quantity and purity by a Nanodrop 1000 spectrophotometer (Isogen, Life Science, Belgium). DNA integrity was assessed by agarose gel-electrophoresis. As previously described [9 (link)], we assessed the within-placental average relative telomere length variation in 14 different placentas and for four different biopsies. As the within placental variation in telomere length was low, i.e. 11.7% we used only one biopsy (1–2 cm³) taken to the right of the main artery for placental telomere length assessment.