Adomas et al. BMC Genomics 2010, 11:477 http://www.biomedcentral.com/1471-2164/11/477 mRNA and the preponderance of RNA in the form of ribosomal (rRNA) or transfer RNA (tRNA). Random hexamers, the most commonly employed, amplify only fraction of the transcriptome, comparing with random pentadecamers [10]. However, random oligonucleotides of any size also prime abundant rRNA and tRNA that can lead to high background and misleading signal. Ribosomal RNA (rRNA) sequences in many prokar- yotes are GC rich relative to the genome at large and are highly conserved. These properties have been used to design non-random hexamers (HD/DHTTTT) to prime reverse transcription reactions [11]. The result was a counter-selective synthesis of cDNA correspond- ing to mRNA from prokaryotic total RNA extractions. In contrast, application of gene-specific primers on a genomic scale requires synthesis of multiple primers. An algorithm to predict the minimal number of non-degen- erate genome-directed primers that specifically anneal to all genes in a given genome has been designed and suc- cessfully applied in bacteria [12]. Another recently developed method relies on a collec- tion of short, computationally selected oligonucleotides (’not-so-random’ (NSR) primers) to obtain full-length, strand-specific representation of nonribosomal RNA transcripts [13]. Selective enrichment of non-rRNA tar- gets was achieved by computationally subtracting rRNA priming sequences from a random hexamer library. The presence of rRNA and tRNA plagues most mRNA puri- fication procedures due to their relative abundance, leading to non-specific interactions like rRNA adsorp- tion to the oligo-dT matrix, or hybridization of rRNA and mRNA sequences [14]. Here we describe an alternate strategy, multi-targeted priming (MTP), that allows for selective amplification of chosen sequences. A degenerate oligonucleotide comple- mentary to selected mRNAs and absent in both rRNA or tRNA was identified allowing for selective transcrip- tion of mRNA. To demonstrate the power of MTP, spe- cies-specific primers were designed and tested on RNA from Saccharomyces cerevisiae exposed to nitrogen defi- ciency, and on RNA from Neurospora crassa during early sexual development following nitrogen depletion. When primary nitrogen sources are not available or are present in concentrations low enough to limit growth, many different nitrogen sources can be used. Utilization of secondary nitrogen sources is highly regulated, and nearly always requires the synthesis of a set of pathway- specific catabolic enzymes and permeases [15]. Several studies have shown the induction of a common suite of effector genes during growth of fungal plant pathogens under nitrogen-starved conditions in vitro and during growth in planta [16,17]. As a consequence, nitrogen- starved media has become a model for the environment that a pathogen encounters during growth in planta Page 2 of 16 [16]. Furthermore, nitrogen uptake and exchange are key processes for ectomycorrhizal interactions that are established between the root systems of terrestrial plants and hyphae from soil-borne fungi [18,19]. Finally, nitro- gen deficiency has been associated with major problems encountered in contemporary wine making [20], espe- cially those related to slow and incomplete fermenta- tions [21]. Therefore, the response of budding yeast exposed to nitrogen starvation has been of interest in light of nutrient depletion during wine fermentation. Neurospora crassa is a heterothallic filamentous fun- gus that undergoes a complex pattern of sexual differen- tiation to form the female reproductive structure (protoperithecium) when subjected to conditions of nitrogen starvation, light, and low temperature [22]. A large number of genes affecting sexual development have been identified by mutation [23,24], but large-scale transcript profiling has not been performed. Here we show for these applications that the addition of an MTP provides superior sensitivity and precision to microarray transcript profiling compared to sole use of oligo-dT primers. We corroborate these results for high throughput RNA tag sequencing, incorporating modifi- cation of the Illumina Digital Gene Expression protocols to facilitate use of MTPs. Results Transcript profiling of S. cerevisiae grown in nitrogen- poor conditions We identified a 12-nucleotide (nt) degenerate sequence that occurs one or more times in 76% of 6608 mRNAs present in budding yeast, Saccharomyces cerevisiae, but that is absent in rRNA or tRNA. The corresponding degenerate primer was NDKTBBBBDWGS. Among the 5039 yeast ORFs containing the identified degenerate sequence, 86% featured one to five exact priming sites and the remaining sequences featured six to 44 priming sites (see Additional File 1). On average, there were 3.2 priming sites per gene. Priming for reverse transcription is not highly specific [8], thus MTPs may prime nearly all genes with some frequency. Nonetheless, the MTP and an equal proportion of oligo-dT primers were sup- plied for the reverse transcription step during the micro- array target labeling procedure, to achieve the greatest coverage possible. Contrasting yeast grown in nitrogen-poor and nitro- gen-rich conditions, priming by MTP increased number of well measured genes, recorded after data normaliza- tion, by 31% over oligo-dT priming (Table 1; see Addi- tional File 2). A logistic regression indicates that a model featuring the number of MTP binding sites in a gene is a statistically significantly better predictor of whether a statistically significant difference in expression will be observed when using MTPs than is a model