Apparently healthy subjects aged 18 to 45 years old, including men and women were recruited and classified into two groups depending on their body mass index (BMI). Normal-weight (NW) group (BMI <25 kg/m2) was composed of 20 subjects and overweight-obese (OW-OB) group (BMI ≥25 kg/m2) of 27 subjects. The inclusion criteria were: subjects with no chronic disease who did not take regular medication or drugs. To avoid potential bias, both groups include approx. 50% men/women and there was no difference in their average age. We recruited 6 additional NW individuals presenting 1 metabolic alteration related to MetS (high plasma total or LDL-cholesterol, plasma triglycerides, or plasma C-reactive protein (CRP) concentrations, or hypertension). They were classified as metabolically obese normal-weight (MONW) individuals. Subjects from the OW-OB group followed a 6-month weight loss program which included a low-calorie food plan (30% reduction in the individual energy requirements) with dietary sessions and exercise counselling. Dietary sessions were offered by a nutritionist every fifteen days who provided face-to-face counselling that was individually adjusted to each subject with the aim of reducing 5% to 10% of its body weight. Neither dietary supplements nor vitamins were provided and all participants consumed self-selected foods. 20 out of the 27 OW-OB subjects who started the study completed the 6-month weight loss program. All the volunteers underwent what we call the fasting test which consisted of collecting blood samples after 4h and after 6 hours after having had a standard breakfast. The blood extractions were performed by skilled health personnel once in the NW and MONW groups, and three times (at the baseline point, and after 3 and 6 months of nutritional intervention) in the OW-OB group. Basic anthropometric parameters were collected: height, weight and waist-hip ratio. Blood pressure was measured with a sphygmomanometer (OMRON HEM705CP, Hoofddorp, The Netherlands) after the volunteer remained seated for fifteen minutes and body composition (fat percentage) was recorded by bioelectrical impedance (OMRON BF306). Additionally, hours of sleep and exercise, as well as food habits (24-h dietary recall) were recorded. Blood extraction was performed after 6h of fast. The following circulating parameters were measured in the analytical service of the Son Espases University Hospital (HUSE): plasma glucose (mg/dL), triglycerides (TG, mg/dL), total cholesterol (mg/dL), high-density lipoprotein cholesterol (HDL-C, mg/dL), gamma-glutamyl transpeptidase (GGT, U/L), and C-reactive protein (CRP, mg/dL). Low-density lipoprotein cholesterol (LDL-C, mg/dL) was calculated using the Friedewald formula whenever TG levels were inferior to 400 mg/dL. Insulin levels (µU/mL) were measured with a commercial ELISA kit of Mercodia (AD bioinstruments, S.L., Terrassa, Spain). Body fat was measured using a DEXA scanner (GE Healthcare/DEXA Lunar Prodigy Primo and Lunar iDEXA; Madison, WI)) connected with an enCore™ software using automatic total body scan mode.
Extracted molecule
total RNA
Extraction protocol
Blood from participants was collected using Vacutainer® EDTA tubes. After blood collection, PBMC were isolated using Ficoll-Paque Plus density gradient media (GE Healthcare Bio Science, Madrid, Spain). In brief, the anticoagulant treated blood was diluted with an equal volume of a phosphate-buffered saline (PBS) solution. Next, the blood was layered carefully over Ficoll without intermixing (10 ml of Ficoll for 20 ml of blood mixed with PBS) in a centrifuge tube and centrifuged at 400 g for 30 min at 20°C. PBMC, together with platelets, were harvested from the interface between Ficoll and plasma layers. This layer was then centrifuged in PBS at 300 g for 10 min at 20°C to wash PBMC and to remove the platelets. PBMC pellet was finally suspended in Tripure Reagent (Roche Diagnostics, Barcelona, Spain) and stored at -80ºC. Total RNA from PBMC samples was extracted using Tripure Reagent (Roche Diagnostics, Barcelona, Spain) and then purified with E.Z.N.A. Total RNA Kit I (Omega Biotek, Vermont, USA), following the manufacturer’s instructions. Finally, RNA was precipitated with isopropanol. Isolated RNA was quantified using a NanoDrop ND 1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). Its integrity was confirmed using agarose gel electrophoresis and the RIN tool using the Agilent 2100 Bioanalyzer System. All samples showed superieor quality and were taken along.
Label
Cy3
Label protocol
100 ng of purified individual total RNA was used for cDNA synthesis, splitted in two equal fractions and used for subsequent cRNA labeling and synthesis using Cy5 and Cy3 dyes and the Agilent low RNA input fluorescent lineair amplification protocol, as described previously by van Schothorst et al. (Anal Biochem 2007;363:315-7). Two samples were taken along as duplicates. Half of the Cy5-labeled samples (40 individual samples, equally spread over all the groups) were used for the reference pool.
Apparently healthy subjects aged 18 to 45 years old, including men and women were recruited and classified into two groups depending on their body mass index (BMI). Normal-weight (NW) group (BMI <25 kg/m2) was composed of 20 subjects and overweight-obese (OW-OB) group (BMI ≥25 kg/m2) of 27 subjects. The inclusion criteria were: subjects with no chronic disease who did not take regular medication or drugs. To avoid potential bias, both groups include approx. 50% men/women and there was no difference in their average age. We recruited 6 additional NW individuals presenting 1 metabolic alteration related to MetS (high plasma total or LDL-cholesterol, plasma triglycerides, or plasma C-reactive protein (CRP) concentrations, or hypertension). They were classified as metabolically obese normal-weight (MONW) individuals. Subjects from the OW-OB group followed a 6-month weight loss program which included a low-calorie food plan (30% reduction in the individual energy requirements) with dietary sessions and exercise counselling. Dietary sessions were offered by a nutritionist every fifteen days who provided face-to-face counselling that was individually adjusted to each subject with the aim of reducing 5% to 10% of its body weight. Neither dietary supplements nor vitamins were provided and all participants consumed self-selected foods. 20 out of the 27 OW-OB subjects who started the study completed the 6-month weight loss program. All the volunteers underwent what we call the fasting test which consisted of collecting blood samples after 4h and after 6 hours after having had a standard breakfast. The blood extractions were performed by skilled health personnel once in the NW and MONW groups, and three times (at the baseline point, and after 3 and 6 months of nutritional intervention) in the OW-OB group. Basic anthropometric parameters were collected: height, weight and waist-hip ratio. Blood pressure was measured with a sphygmomanometer (OMRON HEM705CP, Hoofddorp, The Netherlands) after the volunteer remained seated for fifteen minutes and body composition (fat percentage) was recorded by bioelectrical impedance (OMRON BF306). Additionally, hours of sleep and exercise, as well as food habits (24-h dietary recall) were recorded. Blood extraction was performed after 6h of fast. The following circulating parameters were measured in the analytical service of the Son Espases University Hospital (HUSE): plasma glucose (mg/dL), triglycerides (TG, mg/dL), total cholesterol (mg/dL), high-density lipoprotein cholesterol (HDL-C, mg/dL), gamma-glutamyl transpeptidase (GGT, U/L), and C-reactive protein (CRP, mg/dL). Low-density lipoprotein cholesterol (LDL-C, mg/dL) was calculated using the Friedewald formula whenever TG levels were inferior to 400 mg/dL. Insulin levels (µU/mL) were measured with a commercial ELISA kit of Mercodia (AD bioinstruments, S.L., Terrassa, Spain). Body fat was measured using a DEXA scanner (GE Healthcare/DEXA Lunar Prodigy Primo and Lunar iDEXA; Madison, WI)) connected with an enCore™ software using automatic total body scan mode.
Extracted molecule
total RNA
Extraction protocol
Blood from participants was collected using Vacutainer® EDTA tubes. After blood collection, PBMC were isolated using Ficoll-Paque Plus density gradient media (GE Healthcare Bio Science, Madrid, Spain). In brief, the anticoagulant treated blood was diluted with an equal volume of a phosphate-buffered saline (PBS) solution. Next, the blood was layered carefully over Ficoll without intermixing (10 ml of Ficoll for 20 ml of blood mixed with PBS) in a centrifuge tube and centrifuged at 400 g for 30 min at 20°C. PBMC, together with platelets, were harvested from the interface between Ficoll and plasma layers. This layer was then centrifuged in PBS at 300 g for 10 min at 20°C to wash PBMC and to remove the platelets. PBMC pellet was finally suspended in Tripure Reagent (Roche Diagnostics, Barcelona, Spain) and stored at -80ºC. Total RNA from PBMC samples was extracted using Tripure Reagent (Roche Diagnostics, Barcelona, Spain) and then purified with E.Z.N.A. Total RNA Kit I (Omega Biotek, Vermont, USA), following the manufacturer’s instructions. Finally, RNA was precipitated with isopropanol. Isolated RNA was quantified using a NanoDrop ND 1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). Its integrity was confirmed using agarose gel electrophoresis and the RIN tool using the Agilent 2100 Bioanalyzer System. All samples showed superieor quality and were taken along.
Label
CY5
Label protocol
100 ng of purified individual total RNA was used for cDNA synthesis, splitted in two equal fractions and used for subsequent cRNA labeling and synthesis using Cy5 and Cy3 dyes and the Agilent low RNA input fluorescent lineair amplification protocol, as described previously by van Schothorst et al. (Anal Biochem 2007;363:315-7). Two samples were taken along as duplicates. Half of the Cy5-labeled samples (40 individual samples, equally spread over all the groups) were used for the reference pool.
Hybridization protocol
Individual Cy3-labelled samples were hybridized against the Cy5-labelled reference pool according to the manufacturers' procedure using 2x Hybridization buffer (Agilent). Samples were applied to 8x60K v3 microarrays enclosed in Agilent SureHyb hybridization chambers and hybridized at 65C for 17 hours at 10rpm rotation. After hybridization, slides were washed sequential with GE Wash buffer 2 for 1 minute at 37C, followed by acetonitrile for 1 minute at room temperature, and finally with a stabilization and drying solution for 30 seconds at room temperature according to Agilents' recommendations. Slides were covered by an ozone-barrier slide before scanning.
Scan protocol
Scanned with an Agilent Scanarray scanner (G2505C US22502548) with 10 and 100% laser-power intensities.
Description
Cy5 samples were pooled on a equimolar basis and served as reference pool, and individual Cy3-labelled samples were hybridized against the reference pool.
Data processing
Images were quantified using Agilent Feature Extraction Software (12.2.07) to obtain raw median signal and background values for both Cy3 and Cy5. QC checks were done on raw data and all arrays passed. Spots with a mean signal higher than twice the background value over all arrays and both channels were considered to be expressed. Data was normalized according to Pellis et al. (Physiol Genomics 2003; 16:99-106) based on the Cy5-reference pool, and values were log2 transformed. For final data analysis, 2 duplicate RNA samples were taken along and confirmed for reproducibility but excluded for overall analyses thereafter: US22502548_257236338304_S01_GE2_1200_Dec17_2_2.txt and US22502548_257236338312_S01_GE2_1200_Dec17_1_2.txt.
