A newly designed 45 to 60 mer oligonucleotide Agilent platform microarray for global gene expression studies of Synechocystis
PCC6803: example salt stress experiment
Eneas Aguirre-von-Wobeser 1, Jef Huisman1, Bas Ibelings2 and Hans C.P. Matthijs1
1 Universiteit van Amsterdam, Amsterdam, The Netherlands
2 Netherlands Institute of Ecology, Limnological Institute,
The availability of complete genome sequences makes design of microarrays possible. Different microarray
platforms exist, all of them presenting advantages and disadvantages. Designs may be based on sequenced PCR fragments or alternatively
on in vitro synthesised oligonucleotides. Here we show our oligonucleotide based microarray design for Synechocystis PCC6803.
We designed and implemented a 45 to 60-mer oligonucleotide microarray design for the Agilent microarray synthesis system.
In this work we discuss the design strategy used, which consisted in optimizing several sequence characteristics of the probes.
First, oligonucleotides that had the fewest shared sequences with other parts of the genome were favored. The unique probes
were chosen to be close to 60 bases long, with similar theoretical hybridization strength with their complementary targets,
and avoiding hairpin and dimer structures that could impair hybridization. A trade of between melting temperature and a favourable
low CG content of the probes was a major part of the puzzle to be solved . Three probes per gene have been chosen. The
microarray platform performance was tested with 8 h salt-stressed and control RNA. The observed hybridization intensity was
modestly affected by probe length, uniqueness of sequence, hybridization strength and hairpin-dimer structures. These effects
were accounted for in the calculus design and could be neutralised by normalization. Our results proved internally consistent,
since different parts of genes and genes from probable operons were regulated similarly. The expression changes observed due
to the salt-stress treatment relate largely to those observed in earlier published salt-stress experiments from other laboratories
[2-5]. Some open reading frames showed changes different from those previous observations. The array design, the calculus
methods and the results will be discussed.
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