Technical evaluation of cDNA microarrays.
The aims were to evaluate the common reference design approach in microarray experiments and to evaluate the technical performance and the normalisation of cDNA microarrays with a limited number of spots. Total RNA from 3 normal and 3 tumour sample biopsies were used for synthesis of amino-allyl labelled cRNA. Equal amounts of cRNA from all samples were mixed as reference. After conjugation of cRNA with fluorophores (Cy3/Cy5), each individual tumour cRNA was hybridised to a cDNA microarray together with reference cRNA, scanned and analysed. We show that our procedures for producing cDNA microarrays are reproducible. The concordance between duplicated spots and replicate hybridisation was found to be high. We have demonstrated that our cDNA microarrays are of a high technical quality. The majority of the cDNA microarrays had low local spot background levels. Despite the high background levels for some local spots, variation could be minimized by locally weighted scatter plot smooth normalisation (LOWESS), which we showed was also suitable for normalisation of cDNA microarrays with a limited number of probes.
Frederiksen
Casper M
CM
Molecular Diagnostic Laboratory, Department of Clinical Biochemistry, Aarhus University Hospital, Skejby, DK-8200 Aarhus N, Denmark.
Aaboe
Mads
M
Dyrskjøt
Lars
L
Laurberg
Søren
S
Wolf
Hans
H
Ørntoft
Torben F
TF
Kruhøffer
Mogens
M
D018076:DNA, Complementary; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis; D015203:Reproducibility of Results
D018076:DNA, Complementary