tissue: pooled whole blood RNA of 4 samples developmental stage: children diesease status: healthy
Extracted molecule
total RNA
Extraction protocol
We extracted whole blood RNA using the PAXgene Blood RNA System (Qiagen, Hilden), pooled it in equal amounts to a total of 5 µg from four individuals and synthesized the first- and second-strand cDNA by using the SuperScript Double-Stranded cDNA Synthesis Kit (Life Technologies Inc., Rockville, MD) and an oligo-dT24-T7 primer.
Label
biotin
Label protocol
We prepared cRNA and labeled it with biotinylated UTP and CTP by in vitro transcription using a T7 promoter-coupled double-stranded cDNA as template and the T7 RNA Transcript Labeling Kit (ENZO Diagnostics Inc., Farmingdale, NY).
Hybridization protocol
After purification and precipitation at -20°C, we fragmented 20 µg of this cRNA by heat and ion-mediated hydrolysis at 94°C [200 mM Tris-acetate (pH 8.1), 500 mM KOAc, 150 mM MgOAc] and hybridized it to the microarray. We also added increasing concentrations of spiked controls expected to hybridize to the 5’ and 3’ transcript regions of the Escherichia coli genes bioB, bioC and bioD and of the cre gene of the P1 bacteriophage. We hybridized the arrays with the targets at 45°C for 16 h and then washed them at 25°C with 6 x saline sodium phosphate-EDTA (0.9 M NaCl, 60 mMNaH2PO4, 6 mM EDTA+0.01% Tween 20) for 10 min, followed by a stringent wash at 50°C with 100 mM MES, 0.1 M [Na+], 0.01% Tween 20 for 12 min. We then stained the arrays with 10 μg/ml phycoerythrin-conjugated streptavidin (Molecular Probes Europe BV, Leiden, Netherlands) during 10 min. We did the hybridization, washing and staining procedures using the GeneChip® Fluidics Station 400 (Affymetrix, Santa Clara, CA).
Scan protocol
We determined the fluorescence intensities using a laser confocal scanner (Agilent GeneArray ® Scanner), with excitation 488 nm, spot size 4 µm and pixel space 3µm and analyzed the scanned images using Affymetrix® Microarray Suite version 5.0 (Affymetrix, Santa Clara, CA).
Description
Gene expression data from pooled RNA of whole blood from 4 healthy children
Data processing
We standardized for sample loading and variations in staining by scaling the signals on all arrays to constant target intensity (TGT 150).All microarray results were homogeneous with respect to average background, noise (Raw Q) and percent of genes called present, had spiked control signals within the expected range and adequate 3’/5’ ratio values (under 3) of the signals of the housekeeping gene transcripts (GAPDH, 18SrRNA). The scaling factors of all arrays compared to each other did also not differ by a factor higher than 3.3. These facts allowed us to compare the signals between the microarrays of two groups, totalizing 25 AxCo, 25 UxA, 25 UxCo, 25 SxCo, 25 SxA, 25 SxU, 26 CexCo, 25 CexA, 25 CexU, and 25 SxU comparisons for U133A and U133B (but two U133B microarrays of asymptomatic infection and one of cerebral malaria could not be used due to low hybridization quality, in this case totalizing 15 AxCo and 20 CexCo, CexA, CexU and CexS U133B comparisons). We calculated the signals on the arrays, the signal ratios between them and the corresponding measure of likelihood of change and direction (called “change p-value”) with aid of the Affymetrix® Microarray Suite version 5.0 (Affymetrix, Santa Clara, CA). We excluded probe sets whose signal intensities reached values under 50 in all the investigated groups and considered the expression of a certain gene as increased or decreased if it varied positively by a factor ≥ 1.9 or negatively by a factor ≤ -1.9 and the corresponding change p-value resulted in decreases to lower than 0.15 or in increases to higher than 0.85, respectively, in at least 15 comparisons. We used the one class analysis of the Significance Analysis of Microarrays (SAM) program for the signal ratios (logged on base 2) to identify those genes having significant changes at a false discovery rate of 0.004%.
