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Validation of RNA Arbitrarily Primed PCR Probes Hybridized to Glass cDNA Microarrays: Application to the Analysis of Limited Samples

Institut d’Investigació Biomèdica de Bellvitge (IDIBELL)-Institut Català d’Oncologia,¹ Translational Research Laboratory, Unit of Biostatistics and Bioinformatics, Cancer Epidemiology Department, and ² IDIBELL-Institut de Recerca Oncològica Molecular Oncology Center, L’Hospitalet de Llobregat, Barcelona, Spain.

^aAddress correspondence to this author at: Institut Català d’Oncologia, Laboratori de Recerca Translacional, Av. Gran Via s/n, Km 2.7, 08907 L’Hospitalet de Llobregat, Barcelona, Spain. Fax 34-93-2607466; e-mail gcapella{at}ico.scs.es.

Background: The applicability of microarray-based transcriptome massive analysis is often limited by the need for large amounts of high-quality RNA. RNA arbitrarily primed PCR (RAP-PCR) is an unbiased fingerprinting PCR technique that reduces both the amount of initial material needed and the complexity of the transcriptome. The aim of this study was to evaluate the feasibility of using hybridization of RAP-PCR products as transcriptome representations to analyze differential gene expression in a microarray platform.

Methods: RAP-PCR products obtained from samples with limited availability of biological material, such as experimental metastases, were hybridized to conventional cDNA microarrays. We performed replicates of self-self hybridizations of RAP-PCR products and mathematical modeling to assess reproducibility and sources of variation.

Results: Gene/slide interaction (47.3%) and the PCR reaction (33.8%) accounted for the majority of the variability. From these observations, we designed a protocol using two pools of three independent RAP-PCR reactions coming from two independent reverse transcription reactions hybridized in duplicate and evaluated them in the analyses of paired xenograft-metastases samples. Using this approach, we found that HER2 and MMP7 may be down-regulated during distal dissemination of colorectal tumors.

Conclusion: RAP-PCR glass array hybridization can be used for transcriptome analysis of small samples.

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