HLA-B27/?2-microglobulin transgenic rats on an inbred Fisher 344 background, Gender: Female, Age: 8-10 weeks, Tissue: Colon Mucosa.
Treatment protocol
Colon scrapings were homogenized in liquid N2 using a mortar and pestle cooled with liquid N2.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from the colon homogenates using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Total RNA was purified using Rneasy columns (Qiagen, Venlo, The Netherlands).
Label
Cy5
Label protocol
Amplification and labeling of colonic mucosal samples of individual transgenic rats was performed following the Agilent procedures for whole genome microarray technology (Agilent Technologies, Palo Alto, CA), unless stated otherwise. Briefly, of each animal, 1 ?g total RNA was reverse transcribed using the Agilent Low RNA Input Fluorescent Linear Amplification Kit. Each cDNA sample was split in two fractions which were subsequently used for linear RNA amplification and labeled with Cy5 and Cy3, respectively, using half the amounts as indicated by the manufacturer. The labeled cRNA samples were purified using Rneasy columns (Qiagen).
Channel 2
Source name
Reference RNA. This consists of a pool of equal molar total RNA samples of all groups analyzed.
HLA-B27/?2-microglobulin transgenic rats on an inbred Fisher 344 background, Gender: Female, Age: 8-10 weeks, Tissue: Colon Mucosa.
Treatment protocol
Colon scrapings were homogenized in liquid N2 using a mortar and pestle cooled with liquid N2.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from the colon homogenates using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Total RNA was purified using Rneasy columns (Qiagen, Venlo, The Netherlands).
Label
Cy3
Label protocol
Amplification and labeling of colonic mucosal samples of individual transgenic rats was performed following the Agilent procedures for whole genome microarray technology (Agilent Technologies, Palo Alto, CA), unless stated otherwise. Briefly, of each animal, 1 ?g total RNA was reverse transcribed using the Agilent Low RNA Input Fluorescent Linear Amplification Kit. Each cDNA sample was split in two fractions which were subsequently used for linear RNA amplification and labeled with Cy5 and Cy3, respectively, using half the amounts as indicated by the manufacturer. The labeled cRNA samples were purified using Rneasy columns (Qiagen).
Hybridization protocol
Hybridization was performed by preparing a 2x cRNA target solution containing 825 ng Cy5-labeled cRNA of an individual rat, 825 ng Cy3-labeled pool cRNA, and 11 ?L 10x Blocking agent in a total volume of 52.8 ?L. Fragmentation buffer (2.2 ?L) was added and this was incubated at 65°C for 30 min. Fragmentation was stopped by the addition of 55 ?L 2x GEx hybridization buffer HI-RPM, after which samples were hybridized on 4x 44K rat whole genome Agilent arrays (G4131F) for 17 hours at 65°C in hybridization chambers in a hybridization oven rotating at 10 rpm. After hybridization the arrays were washed according to the manufacturer’s protocol.
Scan protocol
The arrays were scanned with an Agilent G2565B microarray scanner on the basis of integrating both the low and high intensity scans per array, in order to obtain a larger linear range for spot fluorescence quantification according to the manufacturer’s protocol.
Description
Rat Colon Mucosa
Data processing
Signal intensities for each spot were quantified using Feature Extraction 9.5 (Agilent Technologies). 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 1.60 (Applied Maths, Sint-Martens-Latem, Belgium) for analysis. We discarded spots with an average intensity, over all arrays, of Cy5 lower than 2-fold above average background. Then, the Cy5 intensities were normalized as described before (Pellis L, et al. (2003) Physiol Genomics 16: 99-106).
Dietary calcium inhibits colitis development in HLA-B27 transgenic rats
Data table header descriptions
ID_REF
FeatureNum as provided by the manufacturer (Agilent GPL4135)
VALUE
Normalized sample (Cy5) log signal intensity for genes with signal above 2 times background level (blanks are spots below background threshold, which are discarded before normalisation).
