Whole lungs of BALB/c (n=5) exposed to 7 days of 0.5 atm hypoxia and time-matched control mice (n=5) were pooled separately. Total RNA isolation was accomplished with Qiagen RNeasy Midi Kit reagents. Total RNA (50 µg) was annealed with 5 µg oligo dT12-18, and reverse-transcribed into cDNA with Superscript II reverse transcriptase for 2h at 42¡C in the presence of 0.5 mM dGTP, 0.5 mM dCTP, 0.5 mM dATP, 0.3 mM dTTP, 0.2 mM amino-allyl dUTP. After hydrolysis of RNA in 0.2 M NaOH, Tris was removed from the reaction with a Microcon-30 concentrator. The cDNA from Hypoxic and Control groups was covalently coupled separately with Cy5 and Cy3 monoreactive fluors in 50 mM sodium bicarbonate, pH 9.0, followed by quenching with 2.7 M hydroxylamine. The Cy5 and Cy3 labeled cDNAs were combined and purified with a QIAquick PCR purification kit and suspended in 36 µl of 3X SSC and 0.8 mg/ml of poly-A for hybridization to the microarray. Fluorescent array images were collected for both Cy3 and Cy5 with a GenePix 4000A fluorescent scanner and image intensity data were extracted and analyzed with GenePix Pro 4.0 analysis software. After background correction and removal of flagged values, log base 2 expression ratios of median intensities were normalized using lowess method. Keywords = hypoxia, pulmonary hypertension, vascular remodeling
Normalized log2 ratio of background subtracted medians defined by CH2/CH1. Note that this ratio is reversed when a dye switching replication is performed.
CH1_MEDIAN
CH1 (F635) median fluorescence intensity
CH1_MEAN
CH1 (F635) mean fluorescence intensity
CH1_SD
CH1 (F635) fluorescence intensity standard deviation
CH1_BKD_MEDIAN
CH1_BKD_MEAN
CH1 (F635) background mean fluorescence intensity
CH1_BKD_SD
CH2_MEDIAN
CH2 (F532) median fluorescence intensity
CH2_MEAN
CH2 (F532) mean fluorescence intensity
CH2_SD
CH2 (F532) fluorescence intensity standard deviation
CH2_BKD_MEDIAN
CH2 (F532) background median fluorescence intensity
CH2_BKD_MEAN
CH2 (F532) background mean fluorescence intensity
CH2_BKD_SD
CH2 (F532) background fluorescence intensity standard deviation
CH1_MEDIAN - CH1_BKD_MEDIAN
Channel 1 median signal
CH2_MEDIAN - CH2_BKD_MEDIAN
Channel 2 median signal
QUALITY_FLAG
Denotes which features met our filtering criterion. A value of 0 means feature was accepted. A negative value of -50 means that the feature did not have at least 60% of its pixels greater than two standard deviations over the background intensity. A negative value of -100 means that manual inspection of spot revealed unacceptable quality (such as saturation, scratch, etc). Negatively flagged spots were not used in data normalization or VALUE calculation.
