Whole-genome microarrays of fission yeast: characteristics, accuracy, reproducibility, and processing of array data.
BACKGROUND
The genome of the fission yeast Schizosaccharomyces pombe has recently been sequenced, setting the stage for the post-genomic era of this increasingly popular model organism. We have built fission yeast microarrays, optimised protocols to improve array performance, and carried out experiments to assess various characteristics of microarrays.


RESULTS
We designed PCR primers to amplify specific probes (180-500 bp) for all known and predicted fission yeast genes, which are printed in duplicate onto separate regions of glass slides together with control elements (approximately 13,000 spots/slide). Fluorescence signal intensities depended on the size and intragenic position of the array elements, whereas the signal ratios were largely independent of element properties. Only the coding strand is covalently linked to the slides, and our array elements can discriminate transcriptional direction. The microarrays can distinguish sequences with up to 70% identity, above which cross-hybridisation contributes to the signal intensity. We tested the accuracy of signal ratios and measured the reproducibility of array data caused by biological and technical factors. Because the technical variability is lower, it is best to use samples prepared from independent biological experiments to obtain repeated measurements with swapping of fluorochromes to prevent dye bias. We also developed a script that discards unreliable data and performs a normalization to correct spatial artefacts.


CONCLUSIONS
This paper provides data for several microarray properties that are rarely measured. The results define critical parameters for microarray design and experiments and provide a framework to optimise and interpret array data. Our arrays give reproducible and accurate expression ratios with high sensitivity. The scripts for primer design and initial data processing as well as primer sequences and detailed protocols are available from our website.
Lyne
Rachel
R
The Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, UK. rachel@flymine.org
Burns
Gavin
G
Mata
Juan
J
Penkett
Chris J
CJ
Rustici
Gabriella
G
Chen
Dongrong
D
Langford
Cordelia
C
Vetrie
David
D
Bähler
Jürg
J
D017931:DNA Primers; D005456:Fluorescent Dyes; D020869:Gene Expression Profiling / Q000379:methods*; D005800:Genes, Fungal; D023281:Genomics / Q000379:methods; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D015203:Reproducibility of Results; D012568:Schizosaccharomyces / Q000235:genetics* / Q000378:metabolism
D017931:DNA Primers; D005456:Fluorescent Dyes