Long oligonucleotide microarrays in wheat: evaluation of hybridization signal amplification and an oligonucleotide-design computer script.
A computer script was written in the Perl language to design equal-length long oligonucleotides from DNA sequences. The script allows the user to specify G + C content, melting temperature, self-complementarity, the maximum number of contiguous duplicate bases, whether to start with the first start codon and whether to report reverse complements. Microarrays were fabricated with 95 oligonucleotides (60 mers) representing 41 genes. The microarray was interrogated with cDNA from roots and shoots of two near-isogenic lines and a commercial cultivar of Triticum aestivum L. (hexaploid wheat) challenged with cold temperature, hot temperature, or the biological control bacterium Pseudomonas fluorescens. Self-complementarity of the oligonucleotides was negatively correlated with signal intensity in 23 of 54 arrays (39%; P <0.01). Tyramide signal amplification was essential for signal generation and detection. Genes involved in signal transduction pathways responded similarly following exposure to cold, heat and P. fluorescens, suggesting intersection of the pathways involved in response to these disparate stress factors. Microarray results were corroborated by quantitative real-time PCR in 75% of samples assayed. We conclude that long oligonucleotide microarrays for interrogation with cDNA from hexaploid wheat should be constructed from oligonucleotides having minimal self complementarity that also meet user-specified requirements of length, G + C content and melting temperature; multiple oligonucleotides should be used to represent each gene; and Tyramide signal amplification is useful in wheat oligonucleotide microarray studies.
Skinner
Daniel Z
DZ
USDA-ARS and Crop and Soil Science Department, Washington State University, 209 Johnson Hall, Pullman, WA 99164, USA. dzs@wsu.edu
Okubara
Patricia A
PA
Baek
Kwang-Hyun
KH
Call
Douglas R
DR
D001483:Base Sequence; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D012984:Software; D014908:Triticum / Q000235:genetics*