strain: New Zealand white rabbit age: 12 weeks mean weight: 1874 +/- 138 g
Treatment protocol
B : cholesterol rich diet + mix of oleic acid rich oil and tuna oil providing 0.5% of energy as DHA
Growth protocol
New-Zealand white rabbits were fed (7 wk) a high cholesterol diet and received by daily oral gavages either oleic acid rich oil or a mixture of oils providing 0.1% (Group 1), 0.5% (Group 2) or 1% (Group 3) of energy as docosahexaenoic acid. The diet abbreviations T, A, B, C used in the sample records refer to the following : T : cholesterol rich diet + oleic acid rich oil A : cholesterol rich diet + mix of oleic acid rich oil and tuna oil providing 0.1% of energy as DHA B : cholesterol rich diet + mix of oleic acid rich oil and tuna oil providing 0.5% of energy as DHA C : cholesterol rich diet + mix of oleic acid rich oil and tuna oil providing 1% of energy as DHA Diets fed for 7 weeks.
Extracted molecule
total RNA
Extraction protocol
Total RNA from liver tissue was isolated using the Norgen RNA Purification kit (Norgen Bioteck Corporation, Ontario, Canada) according to the manufacturer’s instruction. RNA was quantified with a Nanodrop ND-1000 spectrophotometer (nanoDrop Technologies, Wilmington, Del., USA), and RNA integrity assessed with a RNA 6000 Nano Labchip kit using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, Calif., USA). Only total RNA with an OD 260/230 ratio > 1.7 and a RNA integrity number > 8 was used for microarray hybridization.
Label
Cy3
Label protocol
RNA samples were labelled using the Agilent Quick-Amp Labelling kit (5190-0424) according to the manufacturer’s instructions. Briefly, 500 ng of purified total RNA from each sample was amplified and reverse transcribed in vitro to cDNA using the T7-polymerase, which was subsequently labelled with either cyanine 3-CTP or cyanine 5-CTP dyes (5188-1170-P, Agilent Technologies, Wilmington, USA). The Nanodrop ND-1000 was used to monitor the yield of amplification and the dye incorporation; all samples had a yield > 825ng cRNA and a specific activity > 8.0 pmol Cy3 or Cy5 per ug cRNA.
strain: New Zealand white rabbit age: 12 weeks mean weight: 1874 +/- 138 g
Treatment protocol
T : cholesterol rich diet + oleic acid rich oil
Growth protocol
New-Zealand white rabbits were fed (7 wk) a high cholesterol diet and received by daily oral gavages either oleic acid rich oil or a mixture of oils providing 0.1% (Group 1), 0.5% (Group 2) or 1% (Group 3) of energy as docosahexaenoic acid. The diet abbreviations T, A, B, C used in the sample records refer to the following : T : cholesterol rich diet + oleic acid rich oil A : cholesterol rich diet + mix of oleic acid rich oil and tuna oil providing 0.1% of energy as DHA B : cholesterol rich diet + mix of oleic acid rich oil and tuna oil providing 0.5% of energy as DHA C : cholesterol rich diet + mix of oleic acid rich oil and tuna oil providing 1% of energy as DHA Diets fed for 7 weeks.
Extracted molecule
total RNA
Extraction protocol
Total RNA from liver tissue was isolated using the Norgen RNA Purification kit (Norgen Bioteck Corporation, Ontario, Canada) according to the manufacturer’s instruction. RNA was quantified with a Nanodrop ND-1000 spectrophotometer (nanoDrop Technologies, Wilmington, Del., USA), and RNA integrity assessed with a RNA 6000 Nano Labchip kit using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, Calif., USA). Only total RNA with an OD 260/230 ratio > 1.7 and a RNA integrity number > 8 was used for microarray hybridization.
Label
Cy5
Label protocol
RNA samples were labelled using the Agilent Quick-Amp Labelling kit (5190-0424) according to the manufacturer’s instructions. Briefly, 500 ng of purified total RNA from each sample was amplified and reverse transcribed in vitro to cDNA using the T7-polymerase, which was subsequently labelled with either cyanine 3-CTP or cyanine 5-CTP dyes (5188-1170-P, Agilent Technologies, Wilmington, USA). The Nanodrop ND-1000 was used to monitor the yield of amplification and the dye incorporation; all samples had a yield > 825ng cRNA and a specific activity > 8.0 pmol Cy3 or Cy5 per ug cRNA.
Hybridization protocol
The fluorescently labelled cRNA was hybridized using the Agilent gene expression hybridization kit (5188-5242-A) following the manufacturer’s instructions. Briefly, 825 ng cyanine 3-labelled, linearly amplified cRNA were mixed with 825 ng cyanine 5-labelled, linearly amplified cRNA. The mix was loaded onto the Agilent Rabbit Gene Expression Microarrays (4x44k) following a loop design and hybridization proceeded in a hybridization oven set to 65°C for 17 hours. Then, the slides were washed in solutions I, II and III (Agilent Technologies) and air-dried.
Scan protocol
Slides were scanned immediately after washing using the Agilent DNA Microarray Scanner (G2565AA/G2565BA), at photomultiplier tube voltages red and green. Spot identification and quantification were performed using Agilent Feature Extraction Software 7.0.
Description
Specific peroxidated metabolite issued from LC 3PUFA (4-hydroxyhexenal or 4-HHE) were measured by GC/MS/MS and transcription profiling was conducted in liver
Data processing
Differentially expressed genes were identified using Bioconductor (Moderated p<0.05, Fold Change>1.20) and clustered into pathways (Ingenuity Pathway Analysis 7.0).
