Immediately after blood collection, PBMC were isolated by Ficoll gradient separation.
Growth protocol
Blood samples (1.5-2.5 mL) were collected from rats in feeding conditions from the safena vein, using heparine in NaCl (0.9%) as anticoagulant.
Extracted molecule
total RNA
Extraction protocol
Total RNA from PBMC samples was extracted using Tripure Reagent (Roche Diagnostic Barcelona, Spain) and purified with a Quiagen RNesay Mini Kit spin columns (Izasa SA, Barcelona, Spain).
Label
Cy5
Label protocol
0.5 μg RNA of each sample was reverse transcribed using the Agilent Low RNA Input Fluorescent Linear Amplification Kit (Agilent), according to the manufacturer’s protocol (all materials and reagents are from Agilent Technologies, Palo Alto, CA, USA unless stated otherwise). Half of the cDNA sample (10 µL) was used for linear RNA amplification and labelling with Cy5 or Cy3. All reactions were performed using half of the amounts indicated by the manufacturer. Briefly, a transcription master mix was prepared (7.65 µL nuclease-free water; 10 µL 4x transcription buffer; 3 µL 0.1 M DTT; 4 µL NTP mix, 3.2 µL 50% PEG; 0.25 µL RNaseOUT; 0.3 µL inorganic pyrophosphatase; 0.4 µl T7 RNA Polymerase; 1.2 µL cyanine 3-CTP or cyanine 5-CTP, total volume 30 µL). 30 µL of transcription mastermix was added to10 µL cDNA. In vitro transcription and labelling were carried out at 40°C for 2 h. The labelled cRNA samples were purified using Qiagen Rneasy mini-spin columns (Qiagen, Venlo, The Netherlands). Dye incorporation and cRNA concentration was measured using the ‘micro-array measurement mode’ of the Nanodrop spectrophotometer (NanoDrop Technologies Wilmintog, Delaware, USA). Yield of each individual sample was >825 ng and specific activity >8.0 pmol Cy3 or Cy5 per µg cRNA. All Cy3 cRNAs were pooled to serve as a standard reference pool.
Channel 2
Source name
Reference RNA. This consists of a pool of equal molar total RNA samples of all groups analyzed.
Total RNA from PBMC samples was extracted using Tripure Reagent (Roche Diagnostic Barcelona, Spain) and purified with a Quiagen RNesay Mini Kit spin columns (Izasa SA, Barcelona, Spain).
Label
Cy3
Label protocol
0.5 μg RNA of each sample was reverse transcribed using the Agilent Low RNA Input Fluorescent Linear Amplification Kit (Agilent), according to the manufacturer’s protocol_ch2 (all materials and reagents are from Agilent Technologies, Palo Alto, CA, USA unless stated otherwise). Half of the cDNA sample (10 µL) was used for linear RNA amplification and labelling with Cy5 or Cy3. All reactions were performed using half of the amounts indicated by the manufacturer. Briefly, a transcription master mix was prepared (7.65 µL nuclease-free water; 10 µL 4x transcription buffer; 3 µL 0.1 M DTT; 4 µL NTP mix, 3.2 µL 50% PEG; 0.25 µL RNaseOUT; 0.3 µL inorganic pyrophosphatase; 0.4 µl T7 RNA Polymerase; 1.2 µL cyanine 3-CTP or cyanine 5-CTP, total volume 30 µL). 30 µL of transcription mastermix was added to10 µL cDNA. In vitro transcription and labelling were carried out at 40°C for 2 h. The labelled cRNA samples were purified using Qiagen Rneasy mini-spin columns (Qiagen, Venlo, The Netherlands). Dye incorporation and cRNA concentration was measured using the ‘micro-array measurement mode’ of the Nanodrop spectrophotometer (NanoDrop Technologies Wilmintog, Delaware, USA). Yield of each individual sample was >825 ng and specific activity >8.0 pmol Cy3 or Cy5 per µg cRNA. All Cy3 cRNAs were pooled to serve as a standard reference pool.
Hybridization protocol
Hybridization was performed by preparing a 2x cRNA target solution containing 825 ng Cy5-labeled cRNA, 825 ng Cy3-labeled pool cRNA and 11 µL 10x blocking agent in a total volume of 52.8 µL. Then 2.2 µL fragmentation buffer was added and incubated at 60°C for 30 min. Fragmentation was stopped by adding 55 µL 2x GEx hybridization buffer HI-RPM and hybridized on 4x 44K G4131F whole genome Agilent arrays (Agilent Technologies, Inc. Santa Clara, CA) for 17 h at 65°C in Agilent hybridization chambers in an Agilent hybridization oven rotating at 10 rpm. After hybridization the arrays were subsequently washed with ‘GE wash buffer 1’ for 1 min at room temperature, ‘GE wash buffer 2’ for 1 min at approximately 37°C, acetonitrile for 1 min at room temperature and 30 s at room temperature with ‘stabilization and drying solution’ according to manufacturers protocol (Agilent Technologies).
Scan protocol
Arrays were scanned with an Agilent Microarray Scanner (Agilent Technologies). Spot intensities were quantified using Feature extraction 8.5 (Agilent Technologies).
Description
The data table contains normalized sample (Cy5) signal intensity, and not normalized log ratio (test/reference) data
Data processing
Median density values and background values of each spot were extracted for both the experimental samples (Cy5) and the reference samples (Cy3). Data was exported into GeneMaths XT (Applied Maths, Sint-Martens-Latem, Belgium) for analysis. Spots with an average Cy5 signal intensity, over all arrays, lower than 2-fold average Cy5 local background were discarded. Subsequently, the remaining Cy5 signal intensities were normalized against the Cy3 reference using RIKILT normalization protocol (Pellis L, et al. (2003) Physiol Genomics 16: 99-106).
Diet and feeding condition induced gene expression in rat peripheral blood mononuclear cells
Data table header descriptions
ID_REF
VALUE
Normalized sample (Cy5) signal intensity for genes with signal above 2 times background level (blanks or spots below background threshold, are discarded before normalisation).
