Validation of RNA arbitrarily primed PCR probes hybridized to glass cDNA microarrays: application to the analysis of limited samples.
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.


CONCLUSIONS
RAP-PCR glass array hybridization can be used for transcriptome analysis of small samples.
Grau
Mònica
M
Institut d'Investigació Biomèdica de Bellvitge-Institut Català d'Oncologia, Translational Research Laboratory, Unit of Biostatistics and Bioinformatics, Cancer Epidemiology Department, Barcelona, Spain.
Solé
Xavier
X
Obrador
Antònia
A
Tarafa
Gemma
G
Vendrell
Elisenda
E
Valls
Joan
J
Moreno
Victor
V
Peinado
Miquel A
MA
Capellá
Gabriel
G
D000123:Acetyltransferases / Q000096:biosynthesis / Q000235:genetics; D000818:Animals; D015179:Colorectal Neoplasms / Q000378:metabolism / Q000473:pathology; D018076:DNA, Complementary / Q000737:chemistry*; D005240:Feasibility Studies; D020869:Gene Expression Profiling / Q000379:methods*; D005898:Glass; D051548:Histone Acetyltransferases; D006801:Humans; D020783:Matrix Metalloproteinase 7; D008666:Metalloendopeptidases / Q000096:biosynthesis / Q000235:genetics; D051379:Mice; D008819:Mice, Nude; D008956:Models, Chemical; D009362:Neoplasm Metastasis; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D015347:RNA Probes / Q000737:chemistry*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000096:biosynthesis / Q000235:genetics; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014183:Transplantation, Heterologous
D000123:Acetyltransferases; D018076:DNA, Complementary; D051548:Histone Acetyltransferases; D020783:Matrix Metalloproteinase 7; D008666:Metalloendopeptidases; D015347:RNA Probes; D018719:Erb-b2 Receptor Tyrosine Kinases; C060932:MMP7 protein, human