Assessment of representational difference analysis (RDA) to construct informative cDNA microarrays for gene expression analysis of species with limited transcriptome information, using red and green tomatoes as a model.
Microarray technology makes it feasible to analyse the expression of thousands of different gene elements in a single experiment. Most informative are 'whole genome' arrays, where all gene expression products of a single species or variety are represented. Such arrays are now available for a limited number of model species. However, for other, less well-documented species other routes are still necessary to obtain informative arrays. This includes the use of cDNA libraries. To enhance the amount of information that can be obtained from cDNA libraries, redundancy needs to be minimised, and the number of cDNAs relevant for the conditions of interest needs to be increased. Here, we used representational difference analysis (RDA), a mRNA subtraction procedure, as a tool to enhance the efficiency of cDNA libraries to be used to generate microarrays. Tomato was chosen as a model system for a less well-documented species. cDNA libraries for two distinct physiological conditions of tomato fruits, red and green, were made. The libraries were characterized by sequencing and hybridisation analysis. The RDA procedure was shown to be effective in selecting for genes of relevance for the physiological conditions under investigation, and against constitutively expressed genes. At the same time, redundancy was reduced, but complete normalisation was not obtained, and subsequent sequence analysis will be required to obtain non-redundant arrays. Further, known and putative ripening-related cDNAs were identified in hybridisation experiments on the basis of RNA populations as isolated from the green and red stage of ripening.
Kok
Esther J
EJ
RIKILT Institute for Food Safety, Bornsesteeg 45, 6700 AE Wageningen, The Netherlands. esther.kok@wur.nl
Franssen-van Hal
Nicole L W
NL
Winnubst
Lies N W
LN
Kramer
Evelien H M
EH
Dijksma
Wilko T P
WT
Kuiper
Harry A
HA
Keijer
Jaap
J
D005638:Fruit / Q000378:metabolism*; D020869:Gene Expression Profiling / Q000379:methods*; D015723:Gene Library; D018551:Solanum lycopersicum / Q000235:genetics / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*