  Background: Gene expression studies on non-model organisms require open-end
strategies for transcription profiling. Gel-based analysis of cDNA fragments
allows to detect alterations in gene expression for genes which have neither
been sequenced yet nor are available in cDNA libraries. Commonly used protocols
are cDNA Differential Display (DDRT-PCR) and cDNA-AFLP. Both methods have been
used merely as qualitative gene discovery tools so far. Results: We developed
procedures for the conversion of DDRT-PCR data into quantitative transcription
profiles. Amplified cDNA fragments are separated on a DNA sequencer. Data
processing consists of four steps: (i) cDNA bands in lanes corresponding to
samples treated with the same primer combination are matched in order to
identify fragments originating from the same transcript, (ii) intensity of
bands is determined by densitometry, (iii) densitometric values are normalized,
and (iv) intensity ratio is calculated for each pair of corresponding bands.
Transcription profiles are represented by sets of intensity ratios (control vs.
treatment) for cDNA fragments defined by primer combination and DNA mobility.
We demonstrated the procedure by analyzing DDRT-PCR data on the effect of
secondary metabolites of oilseed rape Brassica napus on the transcriptome of
the pathogenic fungus Leptosphaeria maculans. Conclusion: We developed a data
processing procedure for quantitative analysis of amplified cDNA fragments. The
system utilizes common software and provides an open-end alternative to
microarray analysis. The processing is expected to work equally well with
DDRT-PCR and cDNA-AFLP data and be useful in research on organisms for which
microarray analysis is not available or economical.
