Unknown,Transcriptomics,Genomics,Proteomics

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Integrated molecular portrait of triple negative breast tumors from Mexican women using archived clinical samples

ABSTRACT: Triple negative breast tumours from archived formalin fixed paraffin embeded samples of the National Cancer Institute of Mexico were analyzed for differential gene expressión. Transcriptomic analysis of the 12 tumor samples was done with the FFPE-designed WG-DASL HT assay (Illumina) according to manufacturer’s instructions. This assay measures 29,285 annotated transcripts derived from the RefSeq database corresponding to 20,727 unique genes. Briefly, 200ng of total RNA were reverse-transcribed into biotinylated cDNA, which was then primer-extended with the Assay Specific Oligos. The cDNA was then amplified with universal primers and hybridized to Illumina Human WG DASL HT Expression BeadChip arrays. The Illumina Genome Studio V2010.2 was used to obtain the signal values (AVG-Signal), with no normalization and no background subtraction.The performance of hybridizations was evaluated by assessing the presence of outliers and the noise-to-signal ratios by calculating the ratio of centiles P95/P05 prior to normalisation for each sample. We defined outliers as samples with P95/P05 ratio <9.5. All samples were found to show a correct noise-to-signal ratio (P95/P05>9.6). For differential gene expression analysis, the public dataset GSE32124, which includes 33 fresh frozen tissue samples, generated on the Illumina HumanHT-12 v4.0 beadChip, and which contains 99.98% of the 29,285 probes of the Human WG DASL HT BeadChip was used as normal breast tissue control.

ORGANISM(S): Homo sapiens

SUBMITTER: Magali Olivier

PROVIDER: E-GEOD-62502 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Microarray-based gene expression analysis of peripheral whole blood is a common strategy in the development of clinically relevant biomarker panels for a variety of human diseases. However, the results of such an analysis are often plagued by decreased sensitivity and reliability due to the effects of relatively high levels of globin mRNA in whole blood. Globin reduction assays have been shown to overcome such effects, but they require large amounts of total RNA and may induce distinct gene expression profiles. The Illumina whole-genome DASL (WG-DASL) assay can detect gene expression levels using partially degraded RNA samples and has the potential to detect rare transcripts present in highly heterogeneous whole blood samples without the need for globin reduction. We therefore assessed the utility of the WG-DASL assay in the analysis of peripheral whole blood gene expression profiles. We find that gene expression detection is significantly increased with the use of WG-DASL compared to the standard in vitro transcription-based direct hybridization (IVT), while globin-probe-negative WG-DASL did not exhibit significant improvements over globin-probe-positive WG-DASL. Globin reduction increases the detection sensitivity and reliability of both WG-DASL and IVT with little effect on raw intensity correlations: raw intensity correlations between total RNA and globin-reduced RNA were 0.970 for IVT and 0.981 for WG-DASL. Overall, the detection sensitivity of the WG-DASL assay is higher than the IVT-based direct hybridization assay, with or without globin reduction, and should be considered in conjunction with globin reduction methods for future blood-based gene expression studies. Peripheral whole blood samples were collected from eight human donors in PAXGene tubes. RNA was isolated after freezing and storage, and then prepared for gene expression analysis using the Illumina Human-Ref8 v3.0 BeadChip. Alpha and beta globin were reduced from a portion of the total RNA using the GLOBINclear assay (Ambion, Austin, TX, USA). Two methods of microarray target preparation were examined: Illumina IVT-based direct hybridization (IVT) and Illumina Whole-Genome DASL (WG-DASL). Two DASL Assay Oligo pools (DAP) were utilized for DASL target preparation: the DASL Assay Oligo Pool with globin probes (DAP +) and the DASL Asssay Oligo Pool without globin probes (DAP-).

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Integrated molecular portrait of triple negative breast tumors from Mexican women using archived clinical samples

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