High-throughput screening of suppression subtractive hybridization cDNA libraries using DNA microarray analysis

Abstract

Efficient construction of cDNA libraries enriched for differentially expressed transcripts is an important first step in many biological investigations. We present a quantitative procedure for screening cDNA libraries constructed by suppression subtractive hybridization (SSH). The methodology was applied to two independent SSHs from pearl millet and banana. Following two-colour cyanin dye labelling and hybridization of subtracted tester with either unsubtracted driver or unsubtracted tester cDNAs to the SSH libraries arrayed on glass slides, two values were calculated for each clone, an enrichment ratio 1 (ER1) and an enrichment ratio 2 (ER2). Graphical representation of ER1 and ER2 enabled the identification of clones that were likely to represent up-regulated transcripts. Normalization of each clone by the SSH process was determined from the ER2 values, thereby indicating whether clones represented rare or abundant transcripts. Differential expression of pearl millet and banana clones identified from both libraries by this quantitative approach was verified by inverse Northern blot analysis.