Strain:Swiss Mouse Gender: male Age: 7 weeks Tissue: colon tissue
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from the colon tissues using Trizol (Invitrogen, Carlsbad, CA, USA) as described by the manufacturer, with a subsequent purification step using RNeasy columns (Qiagen, San Diego, CA, USA). RNA was quantified using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) and RNA quality was examined using a RNA 6000 LabChip Kit and a 2100 Bioanalyser (Agilent Technologies, Palo Alto, CA, USA).
Label
cy5
Label protocol
cDNA and cRNA were synthesised using the Low RNA Input Linear Amplification kit (Agilent Technologies, Santa Clara, CA, USA), following the manufacturer’s instructions. Reverse transcription used 500 ng of total RNA and oligo-dT primers. cRNA was synthesised and labeled with either cyanine3 (samples) or cyanine5 (reference) dye (Perkin-Elmer/NEN Life Science, Boston, MA, USA) and the dye incorporation evaluated using a Nanodrop ND-1000 spectrophotometer. Samples ranged from 10 to 22 pmol of dye/µg of RNA.
Strain:mdr1a-/- mouse Gender: male Age: 7 weeks Tissue: colon tissue
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from the colon tissues using Trizol (Invitrogen, Carlsbad, CA, USA) as described by the manufacturer, with a subsequent purification step using RNeasy columns (Qiagen, San Diego, CA, USA). RNA was quantified using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) and RNA quality was examined using a RNA 6000 LabChip Kit and a 2100 Bioanalyser (Agilent Technologies, Palo Alto, CA, USA).
Label
cy3
Label protocol
cDNA and cRNA were synthesised using the Low RNA Input Linear Amplification kit (Agilent Technologies, Santa Clara, CA, USA), following the manufacturer’s instructions. Reverse transcription used 500 ng of total RNA and oligo-dT primers. cRNA was synthesised and labeled with either cyanine3 (samples) or cyanine5 (reference) dye (Perkin-Elmer/NEN Life Science, Boston, MA, USA) and the dye incorporation evaluated using a Nanodrop ND-1000 spectrophotometer. Samples ranged from 10 to 22 pmol of dye/µg of RNA.
Hybridization protocol
Labeled cRNAs were hybridised using an In Situ Hybridization Plus Kit (Agilent Technologies, Santa Clara, CA, USA), following the manufacturer’s protocols, using 750 ng of experimental and reference cRNA onto 44k mouse oligonucleotide arrays (Agilent Technologies, Santa Clara, CA, USA). After hybridisation, slides were washed in solutions I, II and III (Agilent Technologies, Santa Clara, CA, USA) and air-dried.
Scan protocol
Slides were scanned using a GenePix Professional 4200A scanner (Molecular Devices, Sunnyvale, CA, USA) with photomultiplier tube (PMT) voltage of 580 V. Spot identification and quantification was performed using GenePix 6.0 software (Molecular Devices). All slides were individually checked and manually flagged for abnormalities.
Description
MDR1_mice_curcumin_rep4 (Slide 251269421382 High Scan)
Data processing
Microarray data were analysed using the Limma package in Bioconductor. Image analyses of the extracted foreground and background fluorescence intensity measurements were evaluated to select the best background correction for the experiment, but no background correction was necessary due to homogeneous hybridisation. Data were normalised using a local linear regression analysis (LOESS). Data with bad flags (-50 = poor or irregular spots, automatically flagged by the GenePix 4200A scanner; and -100, flagged by hand after visual inspection of the images) were not included in the analysis. Control probes (eQC, Bright corners, E1A and Pro25G) were also removed before normalization. Boxplots, density plots and spatial images of the raw and normalized data were examined in order to check the quality of the microarray data, and that no unusual results for any slide were observed. Data were log transformed before analysis and the mean difference between treatments calculated on this scale, resulting in a log ratio for each probe. The normalized values in the database consist of these log ratios. MA plots of the microarray data were drawn in order to check that there was no dependence of the log ratio on the intensity for any slide. The significance of the log ratio for each probe was determined by calculating one modified t-statistic per probe using an empirical Bayes approach.
The probability values were then corrected for multiple testing using the Benjamini and Hochberg correction, and a false discovery rate (FDR) calculated. Probes that had an FDR of less than 5 % (q<0*05) were considered to be differentially expressed between the groups fed the diets.
