Transcriptome analysis in maritime pine using laser capture microdissection and 454 pyrosequencing

Maritime pine (Pinus pinaster Aiton) is one of the most advanced conifer models for genomics research. Conifer genomes are extremely large and major advances have recently been made in the characterization of transcriptomes. The combination of laser capture microdissection (LCM) and next-generation sequencing is a powerful tool with which to resolve the entire transcriptome of specific cell types and tissues. In the current work, we have developed a protocol for transcriptomic analyses of conifer tissue types using LCM and 454 pyrosequencing. Tissue sections were isolated using non-fixed flash-frozen samples processed by LCM. Complementary DNA synthesis and amplification from tiny amounts of total RNA from LCM samples was performed using an adapted protocol for Conifer RNA Amplification (CRA+). The cDNA amplification yield and cDNA quality provided by CRA+ were adequate for 454 pyrosequencing. Furthermore, read length and quality results of the 454 runs were near the optimal parameters considered by Roche for transcriptome sequencing. Using the CRA+ protocol, non-specific amplifications were prevented, problems derived from poly(A:T) tails in the 454 sequencing technology were reduced, and read length and read number considerably enhanced. This technical approach will facilitate global gene expression analysis in individual tissues of conifers and may also be applied to other plant species.