ArticleJournal of clinical pathology2004
Design of a real time quantitative PCR assay to assess global mRNA amplification of small size specimens for microarray hybridisation.
Article in Journal of clinical pathology, 2004. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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0 citing papers in PubMed, 4 citations in OpenAlex.
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Authors and funding
4 authors at 2 institutions in 1 country.
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No grant is acknowledged in the PubMed record.
Abstract
backgroundLow RNA yields from clinical samples are a limiting step for microarray technology.
aimsTo design an accurate real time quantitative polymerase chain reaction (PCR) assay to assess the crucial step of global mRNA amplification performed before microarray hybridisation, using less than 1 microg total RNA.
methodsThree RNA extraction procedures were compared for small size samples. Total RNA was amplified from universal RNA or the BC-H1 breast cancer micrometastatic cell line using three different protocols. Real time quantitative PCR technology was used for accurate measurement of urokinase plasminogen activator receptor and cytokeratin 8 RNA amplification rates and ratios, using primer sets binding at various distances from the 3' end of transcripts. A 50 mer oligomeric array targeting 87 genes potentially involved in breast cancer metastatic progression was built and hybridised with amplified RNA.
resultsEighteen nanograms of total RNA could be purified from 1000 BC-H1 micrometastatic cells. Amplification rates of 25,000 to 100,000 were achieved with as little as 10 ng of starting material. However, results were highly variable, depending on the amount of starting material, gene characteristics, sample quality, and protocols used. Oligomeric array hybridisation with 20 microg reference RNA resulted in specific and reproducible signals for 83% of the genes, whereas mRNA amplification from less than 400 ng of starting material resulted in selective detection of signals from highly expressed genes.
conclusionsImprovements in the design of global mRNA amplification procedures and oligomeric arrays are needed to extract informative gene expression data from clinical samples containing limited cell numbers.
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