Total RNA samples were extracted from myocardium by Trizol Reagent and purified by RNAesy Columns purification kit (Qiagen) before spectrophotometric quantification and labeling protocols.
Label
Cy5
Label protocol
Briefly, after isolating the polyA-RNA molecules from total RNA by oligo-dT-T7 annealing, complementary double stranded DNA was synthesized and used as template for in vitro transcription by T7 RNA polymerase in presence of biotinilated rUTP. The resulting cRNA was hybridized to slides and finally conjugated with Cy5-streptavidin for detection in laser scanner.
Hybridization protocol
10 micrograms of biotinilated cRNA sample was used per hybridization. The slides were hybridized using the Innova 4080 shaking incubator (Ge Healthcare) for 24 h at 37 C, 300 rpm. After washing, samples were detected by incubation with Cy5-streptavidin at room temperature for 30 minutes, no agitation.
Scan protocol
Following four cycles of 5 min-washes, slides were immediatelly dried by centrifugation at 1000 rpm for 2 min and scanned by Genepix 400B (Axon Instruments).
Description
Biological replicate 1 of 3.
Data processing
The nonlinear intensity-dependent dye bias was normalized intra and inter slides using the Lowess method performed by Codelink package at Bioconductor repository (www.bioconductor.org). Fold changes were calculated by time-paired comparisons among treatment (DMA) samples with the control (DMSO) samples. P-values were generated by performing moderated t-statistic (Lonnstedt and Speed, 2002) on the comparison of normalized data points in the treatment versus the control samples. We selected genes showing significant p-value (< 0.05) and with fold changes > [2.0]. Differentially expressed genes were classified as induced (+) or repressed (-).
Comparison of gene expression induced in hearts excised from mice 24 or 48 hours after DMA or DMSO (vehicle) treatment.
Data table header descriptions
ID_REF
RAW_INTENSITY
value of fluorescence intensity of each spot, non-corrected
NORMALIZED_INTENSITY
value of fluorescence intensity of each spot,corrected (intra-slide correction by median of raw intensities)
QUALITY_FLAG
Numerical Quality score from codes G (good),S (saturated), L (near background), I (irregular), C (contaminated), CI (contaminated and irregular), CL (irregular and near background contaminated)
BKGD_MEAN
average value of fluorescence intensity nearby spot, excluding spot itself
BKGD_MEDIAN
median value of fluorescence intensity nearby spot, excluding spot itself
VALUE
same as UNF_VALUE but with flagged values removed
UNF_VALUE
log2-transformed data, corrected for background contamination and corrected for dye bias (inter-slide correction by lowess adjustment of replicated values)
