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Comparison of expression profiles of once and three time linearly amplified samples

ABSTRACT: In order to assess the impact of three rounds of linear amplification on the technical reproducibility of gene expression measurements, we performed twelve microarray experiments. We analysed mouse RNA from cortex, cerebellum and liver from one individual. One RNA sample of 5µg from each of the three different tissues was processed according to the standard Affymetrix protocol and hybridized onto mouse gene expression arrays MG_U74Av2. Three additional samples from each tissue of 1ng were processed according to a modified procedure that involves three linear amplifications before hybridization onto the microarray chips.

ORGANISM(S): Mus musculus

PROVIDER: GSE21389 | GEO | 2010/05/20

SECONDARY ACCESSION(S): PRJNA126057

REPOSITORIES: GEO

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Project description:Surgically resected gastric adenocarcinoma samples and their paired disease-free (R0) marginal nonmalignant tissue were obtained from 14 patients with gastric adenocarcinoma. RNA was isolated using the RNAeasy Kit (Qiagen Valencia, CA) from 15 mg of tissue. The quality of total RNA and its integrity was assessed using the Bioanalyzer 2100 (Agilent, Palo Alto, CA) and RIN value (RNA Integrity Number) was recorded for all the samples for intact 18S and 28S rRNA. Total RNA (800 ng) from each sample was reverse transcribed and linear amplified using the low RNA input linear amplification kit (Agilent Technologies). After synthesis of the first and second strand of cDNA, the product was used in an in vitro transcription reaction to generate cRNAs in the presence of cyanine 3-labeled UTP (Cy3) for normal and cyanine 5-labeled UTP (Cy5) for tumor. Fragmented Cy3-labeled cRNA of the control sample were mixed with equal amounts of Cy5-labeled cRNA from the gastric tumor sample, and the mixtures were hybridized onto 44K whole human genome DNA microarrays (Agilent Technologies) for 17 hrs at 65°C with constant rotation (10 rpm). Subsequently, the arrays were washed according to manufacturer’s instructions. The slides were scanned using an Agilent microarray scanner (G2565AA), and the images were processed using the Agilent feature extraction software AFE 9.5. GeneSpring GX v10.1 (Agilent Technologies) was used to analyze the expression profiles obtained after microarray hybridization. The feature extraction files from all the samples were uploaded to GeneSpring. Following intensity-dependent normalization (Lowess), the data was subjected to statistical analysis. A two tailed student’s t-test was used to determine significance of the differences observed between the normal and tumor samples. Benjamini Hochberg algorithm was used to compute false discovery rates. Genes were filtered based on P value threshold of 0.001 and false discovery rate of less than 1%.

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Comparison of expression profiles of once and three time linearly amplified samples

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