BMC Genomics 2009, 10:295 http://www.biomedcentral.com/1471-2164/10/295 B. napus seed development. The coexpression of sense and antisense transcripts was confirmed for a subgroup of napin-related transcripts coding for proteins from the large group of protease inhibitor/seed storage/lipid trans- fer protein (LTP) family by strand-specific Real-time RT- PCR. Although Real-time RT-PCR confirms the detection of antisense transcripts that are related to protease inhibi- tor/seed storage/lipid transfer protein (LTP) family pro- teins by LongSAGE, it also indicates that the use of the proteome of A. thaliana for annotation of tag-matched B. napus transcripts in case of diverse gene families limits the resolution and the detailed analysis of the genome and transcriptome structure and availability of more genetic resources for B. napus will help to increase the resolution in the future. The detection of transcripts for a diverse number of genes in antisense orientation by LongSAGE at both time-points suggests strong involvement of antisense transcripts in regulatory processes during B. napus seed development. The comparison of LongSAGE and Real-time RT-PCR data suggests that the sequencing depth applied in this experi- ment limits the LongSAGE detection to transcripts with medium to high copy numbers in B. napus seeds. On the other hand, recent developments in next-generation sequencing (NGS) technologies allow ultra-deep tran- scriptome profiling using transcript tag-based techniques (e.g. DeepSAGE; [31]) that can realistically achieve tran- scriptome saturation and accurate quantification of low- copy transcripts. The data analysis techniques developed in the present study represent a valuable platform for future NGS-based transcriptome tagging studies in Brassica species. As suggested by Shendure [32], such approaches may ultimately replace microarrays as the method of choice for quantitative global transcriptome profiling as a basis for genetical genomics or systems genetics approaches. In the present study we have demon- strated that tag-based transcriptome profiling can also be effectively applied in a non-model, complex polyploid plant species. Conclusion This study underlines the potential of transcript tagging approaches for gene expression profiling in Brassica crop species via EST matching to annotated A. thaliana genes. The data processing pipeline adapted by matching B. napus tags via Brassica ESTs to annotated A. thaliana gene loci enabled differential expression profiling of during seed development in the complex B. napus genome, and furthermore detected an unexpectedly high proportion of EST tags matching in antisense orientation. This suggests a strong involvement of antisense transcript expression in regulatory processes during B. napus seed development. Limits of tag detection for low-abundance transcripts can today be overcome by ultra-high throughput sequencing approaches, so that tag-based gene expression profiling may soon become the method of choice for global expres- sion profiling in non-model species. Methods Plant materials and RNA isolation Plants of the homozygous male-sterile winter oilseed rape maternal line 'MSL-Express' were cultivated under con- trolled growth chamber conditions (16 hour, 20°C day and 8 hour, 16°C night, 60% relative humidity) and man- ually fertilised with pollen from plants of the isogenic male-fertile line 'Express 617'. Pods were harvested weekly from the main racemes of pollinated plants at time-points 14 days up to 70 days after pollination (DAP). The pods were shock-frozen in liquid nitrogen and stored at -80°C, and seeds were collected by splitting the frozen pods. Total RNA was extracted from seeds using RNA Reagent (Invitrogen) and purified from DNA residues by incuba- tion with RQ1 RNAse-free DNAse (Promega), according to the manufacturer's recommendations. LongSAGE library production Total RNA extracted from two seed developmental time- points, 23 DAP and 35 DAP, was processed using the I- SAGELong Kit (Invitrogen) with several modifications to allow more efficient library production and cloning. Briefly, 50 μg of polyA+ RNA was captured on the surface of mag- netic oligo(dT) beads and double stranded cDNA was syn- thesized using SuperScript II reverse transcriptase. Double stranded cDNA was digested for 2.5 hours with NlaIII, and adapters containing MmeI recognition sites were ligated overnight in ligation buffer with 15% polyethylene glycol (PEG) to the 5' end of the cDNAs. Adaptor-linked tags were released from the beads by restriction with MmeI overnight, then precipitated and washed five times with 70% ethanol before being ligated overnight in pairs to form adaptor- linked ditags. These were amplified by PCR and 200 PCR reactions were pooled. Subsequently, 130 bp fragments were purified from 20 lanes on 16% polyacrylamide gels. Purified adapter-linked ditags were redigested overnight with NlaIII to remove adapters and 34 bp ditags were purified from a 16% polyacrylamide gel. The ditags were ligated overnight according to Kenzelmann and Mühlemann [33] to form concatemers, then partially digested with 2 U of NlaIII for 1 min at 37°C according to Gowda et al. [34] to create linear- ized clonable concatemers of suitable sizes. The partially- digested ligation mixture was separated on a 6% polyacryla- mide gel. DNA fragments between 0.8 and 2.5 kb were iso- lated and 1.6 μg concatemers were ligated to 25 ng of linearized pZErO-1 vector digested with SphI. For each of the 23 and 35 DAP libraries more than 3,000 plasmid clones were sequenced in one direction by SeqWright (Houston, TX, USA). SAGE data processing Ditags were extracted from sequence traces using the perl script sage-prhed.pl [35]. Due to the 1% error rate associ- Page 13 of 16 (page number not for citation purposes)