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Sample GSM266997 Query DataSets for GSM266997
Status Public on Feb 21, 2008
Title PMNCs_total RNA Pool_Wash Out_Aliquot N2
Sample type RNA
 
Source name Periferal Blood Mononuclear cells
Organism Homo sapiens
Characteristics total RNA pool
6 males
wash out
AGE: 22-28
3 smokers + 3 non-smokers
BMI: 24.5 ± 3.52 kg/m3
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
 
Submission date Feb 20, 2008
Last update date Feb 20, 2008
Contact name MONTSERRAT FITO COLOMER
E-mail(s) mfito@imim.es
Organization name IMIM
Street address DR.AIGUADER 88
City Barcelona
ZIP/Postal code 08003
Country Spain
 
Platform ID GPL6511
Series (1)
GSE10590 Human in vivo nutrigenomic effect of olive oil

Data table header descriptions
ID_REF
VALUE AB1700 software fully corrected signal intensity
S_N_P/2a-2_OKhymenets_220905

Data table
ID_REF VALUE S_N_P/2a-2_OKhymenets_220905
100002 49787.61 82.1
100003 114.87 -1.06
100027 117.5 -0.64
100036 401.24 1.91
100037 13239.26 35.69
100039 3907.18 22.85
100044 137.13 -0.54
100045 1486.25 5.75
100051 164.21 -3.42
100052 186.62 0.59
100057 1512.73 4.39
100058 44928.84 60
100060 2564.56 14.1
100062 1214.49 2.46
100064 947.93 4.69
100079 5811.8 37.17
100089 1236.28 5.1
100093 203.87 0.16
100095 961.15 -0.57
100100 11663.36 79.72

Total number of rows: 32878

Table truncated, full table size 638 Kbytes.




Supplementary file Size Download File type/resource
GSM266997.txt.gz 1.3 Mb (ftp)(http) TXT
Processed data included within Sample table

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