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Use of non-amplified RNA samples for microarray analysis of gene expression.

ABSTRACT: Demand for high quality gene expression data has driven the development of revolutionary microarray technologies. The quality of the data is affected by the performance of the microarray platform as well as how the nucleic acid targets are prepared. The most common method for target nucleic acid preparation includes in vitro transcription amplification of the sample RNA. Although this method requires a small amount of starting material and is reported to have high reproducibility, there are also technical disadvantages such as amplification bias and the long, laborious protocol. Using RNA derived from human brain, breast and colon, we demonstrate that a non-amplification method, which was previously shown to be inferior, could be transformed to a highly quantitative method with a dynamic range of five orders of magnitude. Furthermore, the correlation coefficient calculated by comparing microarray assays using non-amplified samples with qRT-PCR assays was approximately 0.9, a value much higher than when samples were prepared using amplification methods. Our results were also compared with data from various microarray platforms studied in the MicroArray Quality Control (MAQC) project. In combination with micro-columnar 3D-Gene™ microarray, this non-amplification method is applicable to a variety of genetic analyses, including biomarker screening and diagnostic tests for cancer.

SUBMITTER: Sudo H

PROVIDER: S-EPMC3280296 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Use of non-amplified RNA samples for microarray analysis of gene expression.

Sudo Hiroko H Mizoguchi Atsuko A Kawauchi Junpei J Akiyama Hideo H Takizawa Satoko S

PloS one 20120215 2

Demand for high quality gene expression data has driven the development of revolutionary microarray technologies. The quality of the data is affected by the performance of the microarray platform as well as how the nucleic acid targets are prepared. The most common method for target nucleic acid preparation includes in vitro transcription amplification of the sample RNA. Although this method requires a small amount of starting material and is reported to have high reproducibility, there are also ...[more]

PMID: 22355363

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Project description:Long non-coding RNAs (lncRNAs) serve a crucial role in every aspect of cell biological functions as well as in a variety of diseases, including cardiovascular disease, cancer and nervous system disease. However, the differential expression profiles of lncRNAs in Marfan syndrome (MFS) have not been reported. The aim of the present study was to identify potential target genes behind the pathogenesis of MFS by analyzing microarray profiles of lncRNA in aortic tissues from individuals with MFS and normal aortas (NA). The differentially expressed lncRNA profiles between MFS (n=3) and NA (n=4) tissues were analyzed using microarrays. Bioinformatics analyses were used to further investigate the candidate lncRNAs. Reverse transcription-quantitative (RT-qPCR) was applied to validate the results. In total, the present study identified 294 lncRNAs (245 upregulated and 49 downregulated) and 644 mRNAs (455 upregulated and 189 downregulated) which were differential expressed between MFS and NA tissues (fold change ≥1.5; P<0.05). Gene Ontology enrichment analysis indicated that the differentially expressed mRNAs were involved in cell adhesion, elastic fiber assembly, extracellular matrix (ECM) organization, the response to virus and the inflammatory response. Kyoto Encyclopedia of Gene and Genomes pathway analysis indicated that the differentially expressed mRNAs were mainly associated with focal adhesion, the ECM-receptor interaction, the mitogen-activated protein kinase signaling pathway and the tumor necrosis factor signaling pathway. The lncRNA-mRNA coexpression network analysis further elucidated the interaction between the lncRNAs and mRNAs. A total of five lncRNAs (uc003jka.1, uc003jox.1, X-inactive specific transcript, linc-lysophosphatidic acid receptor 1 and linc-peptidylprolyl isomerase domain and WD repeat containing 1) with the highest degree of coexpression were selected and confirmed using RT-qPCR. In the present study, expression profiles of lncRNA and mRNA in MFS were revealed using microarray analysis. These results provided novel candidates for further investigation of the molecular mechanisms and effective targeted therapies for MFS.

Dataset Information

Use of non-amplified RNA samples for microarray analysis of gene expression.

Publications

Use of non-amplified RNA samples for microarray analysis of gene expression.

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