total RNA pool 6 males wash out AGE: 22-28 3 smokers + 3 non-smokers BMI: 24.5 ± 3.52 kg/m4
Treatment protocol
At first intervention day, at fasting state, 50 mL (44 g) of virgin olive oil were administered in a single dose (experiment 1), following this, volunteers continued with consumption of 25 ml (22 g) of virgin olive oil per day during 3 consequent weeks (experiment 2).
Growth protocol
Prior to the intervention, volunteers followed a one week washout period in which sunflower oil was provided as a source of fat for all purposes. During the first four days of the washout period, participants were asked to follow an antioxidant-controlled diet. Participants were allowed to eat per day less than: 2 pieces of fruit, 2 servings of vegetables or legumes, 2 cups of tea or coffee, and to avoid wine, beer, and olive oil consumption. During the last 3 days before, and on the intervention day, volunteers followed a strict low-phenolic compound diet., according to which several foods, rich in phenolic compounds, were excluded from their diet (vegetables, legumes, fruit, juice, wine, coffee, tea, caffeine-containing soft drinks, beer, cacao, marmalade, olive oil and olives).
Extracted molecule
total RNA
Extraction protocol
Blood was collected at 8 a.m., at fasting state, prior to first (0 hours, pre-dose), 6 h after acute (50 ml (44 g) virgine olive oil) and at 8 a.m., at fasting state, after 3-weeks (25 ml (22 g) virgine olive oil per day) interventions. Periferal Blood Mononuclear Cells (PBMCs) were isolated within two hours after blood drawing using Vacuntainer CPT tubes (Beckton Dickinson, Franklin Lakes, NJ, USA) and following manufacturer´ instructions. Harvested PMNCs were preserved in 1 ml of Ultraspec solution (Biotecx Laboratories, Houston, TX, USA) and were stored at -80ºC prior to RNA extraction. Total RNA was extracted from Ultraspec preserved PMNCs following manufacturer´instructions. RNA concentration (A260) and RNA purity (A260/A280 and A260/A280 ratios) were estimated spectrometrically (NanoDrop® ND-1000, NanoDrop Technologies, USA). RNA integrity was assessed by micro capillary gel electrophoresis (Bioanalyzer, NanoChip, Agilent Technologies, Wilmington, DE, USA) and was estimated by both the rRNA ratio (28S/18S rRNA ratio) and the RIN value (RNA integrity number) using Agilent 2100 Expert Software . The purity of isolated individual RNA samples was greater than 1.8 by A260/A280 and 1.75 by A260/A280, with integrity values not lower than 1.7 and 8.5 by 28S/18S rRNA ratio and by RIN, respectively.The weight equivalents of individual total RNA samples corresponding to baseline, acute and to 3-week intervention were combined into three RNA pooles, respectively. The pooled RNA samples were concentrated using the Rneasy Mini Elute Cleanup system (Qiagen, Barcelona, Spain), till the required by microarray service concentration (0.1 μg/μl). The prepared RNA pools had purity not lower than 1.9 by both A260/A280 and A260/A280 ratios and the integrity was at score of 9.0 according to RIN.The aliquoted pooled and individual RNAs samples were stored at -80º C prior to use.
Label
DIG-UTP
Label protocol
One microgram of total RNA was reverse-transcribed using T7-oligo (dT) primer. After first and second strand cDNA synthesis, the cDNA was purified and used for in vitro transcription (IVT) labeling with DIG-UTP, rendering labeled cRNA, according to the Applied Biosystems instructions for Chemiluminescent RT-IVT labeling kit V.2.0.
Hybridization protocol
Microarrays were prehybridized during 1h at 55ºC. During prehybridization, 10 micrograms of DIG-labeled cRNA were fragmented and used for hybridization (55 ºC, 16 h, 100 rpm agitation), according to Applied Biosystems Chemiluminescence Detection Kit. Same manufacturer´s protocol was used for hybridization washes and for performing Antibody Binding (anti-digoxigenin-AP) and antibody washes. The next steps, chemiluminescent reaction and detection, were performed with only one microarray at a time and following manufactures´ procedures.
Scan protocol
Microarrays were scanned with the Applied Biosystems 1700 Chemiluminescent Analyzer and quantified using the AB1700 software
Description
reference sample
Data processing
The algorithm's quality metrics “flags” and the ratio of signal/noise were used, on each microarray, to quantify probe quality and detectability (1700 Chemiluminescent Microarray Analyzer Users Guide). Probes with a signal/noise ratio <3 and/or flags >5,000 were excluded. From the initial 32,878 probeset, 15,308 high-quality probes were kept, and their signal were normalized among arrays by using the quantile technique
