BMC Genomics 2009, 10:295 http://www.biomedcentral.com/1471-2164/10/295 Comparison of LongSAGE and Real-time RT-PCR analysis A good correlation was observed between expression lev- els estimated by LongSAGE and Real-time RT-PCR for most of the selected genes (Table 3), although some minor differences in estimated fold-change were observed (e.g. for CCR1). This could arise from the more selective specificity of Real-time RT-PCR for transcripts derived from closely-related gene loci, since specific RT-PCR prim- ers were designed from contigs of B. napus ESTs putatively derived from different B. napus gene loci. Furthermore, Real-time RT-PCR may be more sensitive in detecting low abundant transcripts than LongSAGE under the experi- mental conditions and sequencing depth applied in the present study. Transcript diversity Average tag frequencies for the two analysed seed libraries differed significantly (Table 1). Whereas tags with medium frequency (2–20) were more abundant in the 23 DAP library, the 35 DAP library was considerably more enriched for high copy-number tags with a frequency ≥100. Nine unique tags with ≥100 counts each repre- sented 3.2% of the total tag number at 35 DAP, whereas the 8 tags with ≥100 counts each at 23 DAP comprised only 2.1% of the total tags. Accordingly, the relative diver- sity of tags was greater at 23 DAP than at 35 DAP. We detected 48% (7,208 compared to 4,875) more expressed genes at 23 DAP than at 35 DAP (Table 2), which is con- sistent with the number of different tags observed in each of the two libraries. To validate the LongSAGE method and estimate the diver- sity of transcripts, LongSAGE tags derived from selected tags with 2–32 counts in the 35 DAP library were used as primers for RT-PCR in combination with anchored oligo- dT primers. In 17 out of 17 cases a PCR product was amplified and sequenced that aligned in blastn analysis with Brassica or A. thaliana ESTs or cDNAs. A further vali- dation was performed with 9 different LongSAGE tags that had 3 to 325 counts in the 35 DAP library and matched members of the A. thaliana 2S storage gene family or ESTs/ cDNAs from the Brassica napin gene family. In all cases the PCR amplification products, ranging from 75 to 457 bp in length, were related to Brassica napin genes or other protease inhibitor/seed storage/lipid transfer protein (LTP) family protein genes (56 to 100% identity). A high diversity of 3' termini of clones were obtained using the same napin tag primer, with up to 14% divergence, within 200–400 bp of the 3' untranslated region (UTR), from up to 8 clones per amplicon. Interestingly, diverse 3'-UTR Table 3: Comparison of fold-changes for selected genes between 23 and 35 DAP in B. napus seeds measured by LongSAGE and Real- time RT-PCR. A. thaliana gene locus Gene Cumulated counts,35:23 DAP LongSAGE (non-normalized) Fold-change 35:23 DAP LongSAGE (normalized) Fold-change 35:23 B. napus EST DAP Real-time RT- contigs assembled PCR by CAP3 2.0 4.1 2.3 0.9 1.2 1.7 2.1 1224 Contig 1 from 5 ESTs Contig 2 from 2 ESTs 1 EST Contig from 4 ESTs Contig 1 from 22 ESTs Contig 2 from 13 ESTs Contig 3 from 4 ESTs Contig from SAGE primer-amplified cloned 3'-terminal napin gene fragments Page 4 of 16 (page number not for citation purposes) AT2G40890 AT5G48930 AT1G15950 AT2G23910 AT3G19450 AT4G27140 AT4G27150 AT4G27160 AT4G27170 Coumarate 3- hydroxylase (C3H) Hydroxycinnamoyl- Coenzyme A shikimate/quinate hydroxycinnamoyl- transferase (HCT) Cinnamoyl-CoA reductase 1 (CCR1) Cinnamoyl-CoA reductase-related (CCR1-related) Cinnamoyl alcohol dehydrogenase (CAD1) 2S seed storage proteins (napin) 2:2 5:3 2.2:1 0:0 8:9.5 669.1:3.4 560.1:1.9 575.3:1.9 636.5:7.9 1.9 3.2 4.3 - 1.6 374.1 564.4 579.6 155.0 Primers for Real-time RT-PCR were designed using assembled B. napus ESTs matched to A. thaliana proteins using blastx with e-values greater than 1e-6.