Comparative assessment of fungal cellobiohydrolase I richness and composition in cDNA generated using oligo(dT) primers or random hexamers

Understanding soil fungal distribution and activities, particularly at the level of gene expression, is important in unveiling mechanisms regulating their activities in situ. Recent identification of fungal genes involved in carbon cycling has provided the foundation for developing reverse-transcriptase PCR assays to monitor spatiotemporal gene expression patterns in soils and other complex microbial systems. The polyadenylated 3′ ends of eukaryotic mRNA transcripts enables the use of oligo(dT) primers for cDNA synthesis, but this can result in the overrepresentation of the 3′ end of transcripts in cDNA pools. In an effort to increase the uniformity of transcripts represented in cDNA pools, random hexamers have been used. The use of both priming methods is abundant in the literature, but we do not know how these methods perform relative to each other. We performed comparative richness and compositional analyses of the fungal glycosyl hydrolase family 7 cellobiohydrolase I gene cbhI amplified from soil cDNAs that had been generated using either oligo(dT) primers or random hexamers. Our results demonstrate that similar cbhI richness and composition were recovered using both approaches. Richness estimates and compositional profiles of cbhI sequence libraries generated from random hexamer-primed cDNA were more variable than from libraries generated from oligo(dT) primed cDNA. However, our overall results indicate that, on average, comparable richness and composition were recovered from soil cDNAs when either priming method was used.

► The active cellulolytic soil fungal community was assessed by RT-PCR of the cbhI gene. ► Random hexamer and oligo(d)T cDNA synthesis methods from soil RNA were compared. ► Richness and composition of expressed cbhI genes were similar using either method.