tissue: Periferal blood mononuclear cells time: 4 hours after olive oil intake gender: man
Treatment protocol
Prior to the first breakfast intervention, participants followed a 6-week washout period in which they were instructed to not take vitamins, soy supplements, or any drug. To eliminate the potential effect that might exist in their usual dietary habits, all subjects followed a low-fat, carbohydrate rich (CHO) diet during this period and untill the end of the study. In the 24 h before each breakfast intervention, participants were instructed to avoid consuming phenol-rich foods such as fruit or juices, wine, grape juice, chocolate, coffee, tea, olive oil, or soya, and to refrain from intense physical exercise during that period. After a 12 h fasting and following a randomized sequential crossover design with one-week washout period, participants reported to the hospital and received two fat meals consisting of 60 g of white bread, 40 mL of VOO (CANOLIVA®, Antonio Cano e Hijos™, Córdoba, Spain) with high (398 ppm) or low (70 ppm) content in phenolic compounds, and 60,000 IU of vitamin A per m2 of body surface. Olive oil with low content in phenolic compounds was obtained as a result of extraction by physical procedures of most of the phenolic compounds in the high-phenol olive oil, so that both oils kept a similar composition of the remaining macro- and micronutrients.
Growth protocol
Two virgin olive oil-based breakfasts with high (398 ppm) and low (70 ppm) content of phenolic compounds were administered to all the patients following a double-blinded random crossover design in a single dosis two different mornings with a wash-out peridod of one-week between them.
Extracted molecule
total RNA
Extraction protocol
Venous blood samples were obtained at 0 min and 240 min after consumption of each olive oil-based breakfast. Samples from the fasting and postprandial states were collected in tubes containing 1 g EDTA/L and were stored in containers with ice and kept in the dark. Special care was taken to avoid exposure to air, light, and ambient temperature. PBMCs were isolated within 2 h after blood draw from 30 mL EDTA anticoagulated blood samples taken 4 hours after the consumption of each olive oil-based breakfast. Buffy coats were diluted 1:2 in PBS, and cells were separated in 5 mL Ficoll gradient (lymphocyte isolation solution, Rafer) by centrifugation at 2000 x g for 30 min. PBMCs were collected and washed twice with cold PBS. Harvested PBMCs were preserved in liquid nitrogen and stored at -80ºC prior to RNA extraction. Total RNA was extracted from mononuclear cells by using of TRI Reagent (Sigma-Aldrich, Inc., St. Louis, MO, USA) and then cleanup by RNeasy MiniElute Cleanup Kit (Qiagen, Hilden, Germany). Recovered RNA was quantified using a Nanodrop ND-1000 v3.5.2 spectrophotometer (Nanodrop Technology, Cambridge, UK). RNA integrity was assessed using 1.6% agarose gel, TBE 1x. RNA was judged as suitable for array hybridization only if samples exhibited intact bands corresponding to 18S and 28S ribosomal RNA subunits.
Label
Cy5
Label protocol
RNA samples were labeled using the SuperScript Indirect RNA Amplification System (Invitrogen Inc., Carlsbad, CA, USA) according to the manufacturer's instructions. To avoid confounding by extraneous factors, all the experiments were performed in a single batch and processed by one researcher on the same day for each step. Briefly, 1 μg of total RNA was reverse transcribed using SuperScript III Reverse Transcriptase and second strand synthesized by the DNA Polymerase I and DNA Ligase. Amplification and amino-allyl labeling were performed by T7 RNA polymerase in vitro transcription, to give amino-allyl UTP labeled RNA (aRNA). aRNA samples from PBMCs obtained after the intake of high and low phenol olive oils were labeled with Alexa Fluor 555 and Alexa Fluor 647 dyes (Invitrogen Inc., Carlsbad, CA, USA). The dye incorporation rate was assessed with a Nanodrop ND-1000 v3.5.2 spectrophotometer (Nanodrop Technology, Cambridge, UK) and was found to be between 8 and 12 pmol.μL-1.
tissue: Periferal blood mononuclear cells time: 4 hours after olive oil intake gender: man
Treatment protocol
Prior to the first breakfast intervention, participants followed a 6-week washout period in which they were instructed to not take vitamins, soy supplements, or any drug. To eliminate the potential effect that might exist in their usual dietary habits, all subjects followed a low-fat, carbohydrate rich (CHO) diet during this period and untill the end of the study. In the 24 h before each breakfast intervention, participants were instructed to avoid consuming phenol-rich foods such as fruit or juices, wine, grape juice, chocolate, coffee, tea, olive oil, or soya, and to refrain from intense physical exercise during that period. After a 12 h fasting and following a randomized sequential crossover design with one-week washout period, participants reported to the hospital and received two fat meals consisting of 60 g of white bread, 40 mL of VOO (CANOLIVA®, Antonio Cano e Hijos™, Córdoba, Spain) with high (398 ppm) or low (70 ppm) content in phenolic compounds, and 60,000 IU of vitamin A per m2 of body surface. Olive oil with low content in phenolic compounds was obtained as a result of extraction by physical procedures of most of the phenolic compounds in the high-phenol olive oil, so that both oils kept a similar composition of the remaining macro- and micronutrients.
Growth protocol
Two virgin olive oil-based breakfasts with high (398 ppm) and low (70 ppm) content of phenolic compounds were administered to all the patients following a double-blinded random crossover design in a single dosis two different mornings with a wash-out peridod of one-week between them.
Extracted molecule
total RNA
Extraction protocol
Venous blood samples were obtained at 0 min and 240 min after consumption of each olive oil-based breakfast. Samples from the fasting and postprandial states were collected in tubes containing 1 g EDTA/L and were stored in containers with ice and kept in the dark. Special care was taken to avoid exposure to air, light, and ambient temperature. PBMCs were isolated within 2 h after blood draw from 30 mL EDTA anticoagulated blood samples taken 4 hours after the consumption of each olive oil-based breakfast. Buffy coats were diluted 1:2 in PBS, and cells were separated in 5 mL Ficoll gradient (lymphocyte isolation solution, Rafer) by centrifugation at 2000 x g for 30 min. PBMCs were collected and washed twice with cold PBS. Harvested PBMCs were preserved in liquid nitrogen and stored at -80ºC prior to RNA extraction. Total RNA was extracted from mononuclear cells by using of TRI Reagent (Sigma-Aldrich, Inc., St. Louis, MO, USA) and then cleanup by RNeasy MiniElute Cleanup Kit (Qiagen, Hilden, Germany). Recovered RNA was quantified using a Nanodrop ND-1000 v3.5.2 spectrophotometer (Nanodrop Technology, Cambridge, UK). RNA integrity was assessed using 1.6% agarose gel, TBE 1x. RNA was judged as suitable for array hybridization only if samples exhibited intact bands corresponding to 18S and 28S ribosomal RNA subunits.
Label
Cy3
Label protocol
RNA samples were labeled using the SuperScript Indirect RNA Amplification System (Invitrogen Inc., Carlsbad, CA, USA) according to the manufacturer's instructions. To avoid confounding by extraneous factors, all the experiments were performed in a single batch and processed by one researcher on the same day for each step. Briefly, 1 μg of total RNA was reverse transcribed using SuperScript III Reverse Transcriptase and second strand synthesized by the DNA Polymerase I and DNA Ligase. Amplification and amino-allyl labeling were performed by T7 RNA polymerase in vitro transcription, to give amino-allyl UTP labeled RNA (aRNA). aRNA samples from PBMCs obtained after the intake of high and low phenol olive oils were labeled with Alexa Fluor 555 and Alexa Fluor 647 dyes (Invitrogen Inc., Carlsbad, CA, USA). The dye incorporation rate was assessed with a Nanodrop ND-1000 v3.5.2 spectrophotometer (Nanodrop Technology, Cambridge, UK) and was found to be between 8 and 12 pmol.μL-1.
Hybridization protocol
Differentially labeled aRNA samples were cohybridized on microarray slides. Hybridization was carried out using the Gene Expression Hybridization Kit (Agilent Technologies Inc., Santa Clara, CA, USA) following the manufacturer's instructions. Briefly, 825 ng of each fluorescent-labeled aRNA sample was mixed with blocking reagent and fragmentation buffer and incubated 30 min at 60º. Hybridization was done at 65°C for 17 h at continuous rotation of the hybridization chambers according to the Agilent manual. Slides were disassembled and washed in solutions I and II, and dried using a nitrogen-filled air gun before scanning.
Scan protocol
Microarray images were obtained by scanning each slide using a Gene Pix 4000B scanner (Axon Instruments, Union City, CA). Image quantitation was performed using Agilent Feature Extraction Software v9.5 (Agilent Technologies Inc., Santa Clara, CA, USA).
Description
Sub#17_rep_1_dye swap.txt
Data processing
For the Agilent oligonucleotide arrays, the background adjusted signals (BGSubSignal) calculated by the Agilent Feature Extraction Software (v9.5) were used, filtered on the flag IsWellAboveBG. Log2 dye swaped (red/green) mean-centered signal ratio data were within-array and between-array normalized by lowess and quantile methods respectively, both implemented in the R Limma package (GLP).
