Adomas et al. BMC Genomics 2010, 11:477 http://www.biomedcentral.com/1471-2164/11/477 Page 9 of 16 Table 3 Overview of transcriptomic sequence reads, comparing numbers of matches to coding ORFs, rRNAs, and tRNAs Oligo-dT N-rich Oligo-dT N-poor MTP N-rich MTP N-poor Single match mRNA 266258 569310 1286862 1444282 Single match rRNA 40 38 438 1418 Single match tRNA 18 12 18 21 Multiple matches 72849 99222 1197105 470558 Unmatched 258323 673334 1312253 1136358 expressed in N. crassa protoperithecia than were abun- dantly expressed (Figure 1b). While the functional clas- sifications with the highest number of genes significantly differentially expressed in protoperithecia were the “unknown” functional category or were metabolism- related, the oligo-dT-primed experiment indicated that genes involved in protein synthesis were significantly affected by sexual development (Fisher’s Exact test, P = 2 × 10-6). Application of MTPs for N. crassa microarray profiling additionally identified the functional categories of metabolism (P = 0.003) and energy (P = 0.03) as including a higher proportion of genes differentially expressed in protoperithecia. Enzymes involved in fatty acid metabolism were frequently abundantly expressed in N. crassa protoperithecia as compared to mycelia (Figure 5), while genes coding for ribosomal proteins were generally meagerly expressed (see Additional File 7). Microarray hybridizations performed with MTP- based reverse transcription identified 1 to 12 more genes involved in each biological process than did microarrays performed using oligo-dT primers only (Table 2). MTPs outperformed oligo-dT primers most strikingly in their ability to reveal differentially expressed genes in the category of unknown and unclassified pro- teins (Figure 1b). A gene involved in control of sexual development, encoding clock controlled pheromone ccg- 4 precursor, was among genes differentially expressed in N. crassa protoperithecia (see Additional File 6). Table 4 Overview of differential expression from transcriptomic tag sequencing, comparing results of oligo-dT and multi-targeted primers (MTPs) for reverse transcription Feature Recorded genes Genes significantly differentially expressed (P ≤ 0.05) Up-regulated genes (P ≤ 0.05) Down-regulated genes (P ≤ 0.05) Highest gene expression ratio Average gene expression ratio Saccharomyces cerevisiae Nitrogen depletion MTP Common Oligo (dT) 4858 2272 958 1314 166 1.84 5078 2861 1437 1427 284 1.90 4710 1437 522 633 Discussion Our multi-targeted priming method features selective targeting of multiple genomic sequences and counter- selection against undesirable sequences. For genome- wide gene expression assays, the addition of multi- targeted primers offers the potential for improving the coverage of the reverse transcription. Reverse transcrip- tion reactions are fairly nonspecific: transcription may be primed by non-complementary primers, other RNA molecules present in the sample, and even by dNTPs [8], so the priming strategy selected may be expected to play a significant role in determining the results obtained. For instance, commonly-used oligo-dT pri- mers generate a high frequency of truncated cDNAs through internal poly-A priming [26]. We demonstrated that addition of MTP to commonly used oligo-dT primers for yeast and N. crassa microarray profiling sub- stantially increased the number of well measured genes recorded after data normalization as well as the number of genes significantly differentially expressed, compared with the sole use of oligo-dT primers (Table 1). Addi- tionally, MTP-based profiling manifested higher statisti- cal power as demonstrated by lower value of GEL50, a measure of empirical power to reveal differences in gene expression [27], in the S. cerevisiae experiment. High-throughput RNA tag sequencing validated our findings. It confirmed the utility of MTPs, combined with oligo-dT column purification, for priming reverse transcription reactions, yielding an increased number of detected and well measured genes, compared to oligo- dT primers alone (Table 3). Priming using MTPs, we were able to corroborate gene expression levels for a higher number of genes than when priming with oligo- dT (41% and 35%, respectively; Figures 2, 3, 4). To explain this result, note that both traditional oligo-dT and MTP priming provide biased counts and profile only a proportion of the total RNA pool. Some genes that are poorly primed by oligo-dT primers are those that have protected poly-A tails due to foldback struc- tures; other genes, such as histones in metazoan DNA, have no poly-A tail. In a particuliarly clear case for yeast, it has been demonstrated that many to all of the 24 putative and known mitochondrial mRNAs are not stabilized by a poly-A tail [28-31]. For these genes, MTPs were successful at priming and revealing gene