tissue: whole blood time point: after twelve weeks (t12) of supplementation race: Caucasian gender: male age: between 29 and 51 years condition: dyslipidemic (TC > 200 mg/dl; LDL > 130 mg/dl; TG > 150 mg /dl) supplementation: corn oil capsules (3.05 g linoleic acid per day)
Treatment protocol
Venous blood samples were collected into PAXgene Blood RNA Tubes (PreAnalytiX, Hombrechtikon, Switzerland) at baseline (t0), 4 hours (t4h) after the first intake of the study products, after one week (t1), and after twelve weeks (t12) of supplementation. The collected whole blood samples were incubated for 24 hours in the PAXgene Blood RNA Tubes at room temperature. The patients gave informed consent.
Extracted molecule
total RNA
Extraction protocol
Total RNA from whole blood was isolated using the PAXgene Blood RNA Kit (Qiagen, Hilden, Germany) according to the manufacturer's recommended procedures. Afterwards, total RNA was purified with the Globin Clear Kit (Ambion, Applied Biosystems, Darmstadt, Germany) according to the manufacturer's instructions. Equal amounts of purified RNA samples from each member of the respective group were pooled together. This was done for all different time points (t0, t4h, t1 and t12). Therefore, four pool-samples were generated by this process for each group, which results in a total of sixteen pool-samples for the microarray experiments. This approach was chosen to reduce biological inter-individual variability, which is frequent due to the variations in relative proportions of specific blood cell subsets, gender, age, and disease state.
Label
fluorescien
Label protocol
During reverse transcription, 6 µg of purified total RNA was converted into fluorescein (Fl) and Biotin (B) labeled cDNA and hybridized simultaneously in one experiment to the same array. After hybridisation, FL- and B-labeled cDNAs were sequentially detected with a series of conjugate reporter molecules according to the TSA process, ultimately with Tyramide-Cy3 and Tyramide-Cy5, respectively.
tissue: whole blood time point: baseline (t0) race: Caucasian gender: male age: between 29 and 51 years condition: dyslipidemic (TC > 200 mg/dl; LDL > 130 mg/dl; TG > 150 mg /dl) supplementation: corn oil capsules (3.05 g linoleic acid per day)
Treatment protocol
Venous blood samples were collected into PAXgene Blood RNA Tubes (PreAnalytiX, Hombrechtikon, Switzerland) at baseline (t0), 4 hours (t4h) after the first intake of the study products, after one week (t1), and after twelve weeks (t12) of supplementation. The collected whole blood samples were incubated for 24 hours in the PAXgene Blood RNA Tubes at room temperature. The patients gave informed consent.
Extracted molecule
total RNA
Extraction protocol
Total RNA from whole blood was isolated using the PAXgene Blood RNA Kit (Qiagen, Hilden, Germany) according to the manufacturer's recommended procedures. Afterwards, total RNA was purified with the Globin Clear Kit (Ambion, Applied Biosystems, Darmstadt, Germany) according to the manufacturer's instructions. Equal amounts of purified RNA samples from each member of the respective group were pooled together. This was done for all different time points (t0, t4h, t1 and t12). Therefore, four pool-samples were generated by this process for each group, which results in a total of sixteen pool-samples for the microarray experiments. This approach was chosen to reduce biological inter-individual variability, which is frequent due to the variations in relative proportions of specific blood cell subsets, gender, age, and disease state.
Label
biotin
Label protocol
During reverse transcription, 6 µg of purified total RNA was converted into fluorescein (Fl) and Biotin (B) labeled cDNA and hybridized simultaneously in one experiment to the same array. After hybridisation, FL- and B-labeled cDNAs were sequentially detected with a series of conjugate reporter molecules according to the TSA process, ultimately with Tyramide-Cy3 and Tyramide-Cy5, respectively.
Hybridization protocol
Slides were incubated at 42°C for 120 min in the following preheated (42°C) solution: 5xSSPE, SDS 0.1%, BSA 1%. Slides were washed for 2 minutes in deionized water followed by a centrifugation for drying. Equal amounts of biotin-labeled cDNA and fluorescein-labeled cDNA were hybridized simultaneously in one experiment to the human whole genome OneArray™ Microarray (Phalanx Biotech Group; Belmont, CA, USA). Hybridizations were carried out overnight at 42 °C in hybridization chambers (Eppendorf AG, Hamburg, Germany). After hybridization of the samples on the microarray slides overnight, slides were washed for 5 minutes with 2xSSCP, 5 minutes with 1xSSCP and 5 minutes with 0,5xSSCP at room temperature.
Scan protocol
The hybridized chips were scanned at least six times with an Axon 4000 B™ scanner at different settings, both changing the PMT and the laser power settings. Expression levels were quantified using GenePix Pro 6.0™ image analysis software (Axon Instruments, CA, USA). Artifact-associated spots were eliminated by both visual and software guided flags.
Description
The human OneArray™ DNA microarray is made of sense-strand 60-mer polynucleotide probes spotted onto a proprietary chemical layer-coated microarray glass slide. Each probe is spotted onto the array using a non-contact spotting technology. Each microarray contains 30,968 human genome probes (Phalanx Biotech Group, Palo Alto, CA 94304-1124, USA).
Data processing
A ratio-based normalization method was carried out. A locally weighted linear regression (Lowess) was employed as a normalization method to account for intensity-dependent effects. The mean of the data for differently labelled targets for each gene on respectively two microarrays was taken. A standard two-state pooled-variance t-test (5% probability of error) was applied to detect differentially expressed genes. VALUE is the normalized Log (base 2) ratio of the median of Channel 1 to Channel 2.
The multiple gpr files per Sample represent the multiple scans using different settings.
