A common reference for cDNA microarray hybridizations

Center for Human and Clinical Genetics, Leiden University Medical Center, Wassenaarseweg 72, 2333AL Leiden, Nederland.
Nucleic Acids Research (Impact Factor: 9.11). 12/2002; 30(21):e116.
Source: PubMed


Comparisons of expression levels across different cDNA microarray experiments are easier when a common reference is co-hybridized to every microarray. Often this reference consists of one experimental control sample, a pool of cell lines or a mix of all samples to be analyzed. We have developed an alternative common reference consisting of a mix of the products that are spotted on the array. Pooling part of the cDNA PCR products before they are printed and their subsequent amplification towards either sense or antisense cRNA provides an excellent common reference. Our results show that this reference yields a reproducible hybridization signal in 99.5% of the cDNA probes spotted on the array. Accordingly, a ratio can be calculated for every spot, and expression levels across different hybridizations can be compared. In dye-swap experiments this reference shows no significant ratio differences, with 95% of the spots within an interval of +/-0.2-fold change. The described method can be used in hybridizations with both amplified and non-amplified targets, is time saving and provides a constant batch of common reference that lasts for thousands of hybridizations.

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Available from: Judith M Boer, Feb 07, 2014
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    • "Therefore, microarray hybridisations were performed for each treatment (control, WC 30 μg/ml, WC-Co 33 μg/ml, CoCl2 3 μg/ml; 3 h and 3 d exposure each) with 5 independent biological replicates. All hybridisations were performed against a common reference RNA [72] consisting of a mixture of equal amounts of RNA from all treatments. Synthesis of cDNA, cRNA and cRNA-labeling was performed with the Agilent Low RNA Input Linear Amplification Kit according to the manufacturer's instructions. "
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    • "One sample is prepared from a reference mRNA and the other from mRNA isolated from the experimental cells. The common reference, or universal control, is collected from a pool of cell lines or a mix of all analysed samples [26] [9]. The initial data arising from cDNA microarray experiments are relative mRNA levels – experiment to control ratios. "
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    • "Relating each experimental sample to a common reference standard facilitates the comparison of ratios across datasets [4]. Several types of reference samples based on commercial Universal reference RNA [5], genomic DNA [6-8] or PCR products representing the collection of cDNA clones printed on the chip [9,10] have been proposed, but no single universal reference standard is widely adopted, seriously impeding cross comparisons between different studies. The composition and properties of the selected reference sample must be addressed properly, because it raises issues concerning the experimental design, the goal of the study and the long term comparability of the data. "
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