Two color low input quick amp labeling kit

Fifty ng of total RNA extracted were amplified and fluorescently labeled to produce Cy5 or Cy3 cRNA using the Low Input Quick Amp Labeling two color Kit (Agilent, Santa Clara, CA, United States) in the presence of spike-in two color control, as recommended by the manufacturer. Following the purification, 825 ng of labeled cRNA were hybridized onto G4845A Human GE 4 × 44K v2 microarray (Agilent, Santa Clara, CA, United States) containing 27958 Entrez Gene RNAs sequences. Microarrays were scanned with Agilent G2505 scanner (Agilent, Santa Clara, CA, United States) and data extracted by Feature Extraction software (Agilent, Santa Clara, CA, United States) version 11.0 using linear and Lowess normalization. Statistical analyses were performed using the free R 2.1 software (http://www.r-project.org). Data were analyzed by Student’s t-test to detect differentially expressed genes, and the probability values were adjusted by Benjamini-Hochberg correction for multiple testing at 0.1 to eliminate false positives.

Total RNA (50 ng per sample) was amplified and fluorescently labeled to produce Cy5 or Cy3 complementary RNA (cRNA) by using the Low Input Quick Amp Labeling Two-Color Kit (Agilent, Santa Clara, CA, USA) in the presence of a two-color spike-in control, as recommended by the manufacturer. After purification, 825 ng of labeled cRNA was hybridized onto G4845A Human GE 4x44K v2 microarray (Agilent, Santa Clara, CA, USA) according to the manufacturer’s instructions. The G4845A Human GE 4x44K v2 microarray contains 27,958 Entrez Gene RNA sequences. After hybridization, an Agilent G2505 scanner (Agilent, Santa Clara, CA, USA) was used to scan microarrays. The hybridization data were extracted by means of the Feature Extraction software version 11.0 and analyzed through the GeneSpring GX software version 14.5 (Agilent Technologies, Santa Clara, CA, USA). Data were normalized using 50th percentile shift and analyzed with moderated t-tests corrected by Westfall–Young permutation with corrected p-value cut-off set to 0.05. All transcripts presenting p < 0.05 were considered differently expressed.

Fifty ng of total RNA extracted from each sample were amplified and fluorescently labelled to produce Cy5 or Cy3 cRNA using the Low Input Quick Amp Labeling two-color kit (Agilent, USA) in the presence of spike-in two colours control. After purification, 825 ng of labelled cRNA/sample were hybridized onto G4845A Human GE 4x44K v2 microarrays (Agilent, USA) according to the manufacturer's instructions. The G4845A Human GE 4x44K v2 microarray contains 27,958 Entrez Gene RNAs sequences. After hybridization, microarrays were scanned with Agilent G2505 scanner (Agilent, USA) and data were extracted with Feature Extraction software (Agilent, USA) using Lowess normalization. Statistical analyses were performed using the free R 2.1 software (http://www.r-project.org) after controlling the variance of each gene. Data were analyzed with a Student's t test to detect differentially expressed genes and the probability values were adjusted using the Benjamini-Hochberg correction for multiple testing at 0.1 to eliminate false positives. Genes selected by these criteria are referred to as the “differentially expressed genes”. Microarray data have been deposited in accordance to the MIAME guidelines in the public Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo/), accession number GSE54325 and GSE54643.