Adomas et al. BMC Genomics 2010, 11:477 http://www.biomedcentral.com/1471-2164/11/477 Page 8 of 16 Figure 4 Genes significantly differentially expressed (P ≤ 0.05) by S. cerevisiae growing in nitrogen-poor (light red/blue) and nitrogen- rich (dark red/blue) conditions coding for enzymes involved in citrate cycle (TCA); identified by microarray using multi-targeted primers (red) or oligo(dT) primers (blue). The results were validated using RNA sequencing on Illumina platform (grey; bars in the same order as for microarray profiling). Error bars represent 95% credible intervals. NS - statistically insignificant difference (P > 0.05); * or lack of a symbol - significant difference (P ≤ 0.05); † - not well measured. was reverse complementary to 12-nt sequences found one or more times in 85% of the predicted mRNAs, one time in one tRNA, and one time in one rRNA. The MTP was incorporated into an experiment to quantify gene expression in mycelia and protoperithecia formed by the fungus during growth on nitrogen-poor medium, and the results were compared with transcript profiling using solely oligo-dT-based reverse transcription. Appli- cation of multi-targeted primers increased the number of well-measured genes by 331% (Table 1, see Addi- tional File 6). The number of genes identified as signifi- cantly differentially expressed in N. crassa protoperithecia rose by 136%. The highest and the aver- age gene expression ratio estimated based on the MTP- based experiment were lower than for data obtained with only oligo-dT primers (Table 1). The GEL50 value was similar when MTPs were included, compared to when only oligo-dT primers were included. However, 38% more genes were detected as differentially expressed in the MTP experiment above the GEL50 threshold than were differentially expressed above the GEL50 threshold in the experiment primed with oligo-dT primers alone (Table 1). Thus, more gene expression levels were precisely estimated with MTPs, even though in this case there was not a finer level of resolution for small changes in gene expression. Functional classification of genes significantly differentially expressed by N. crassa protoperithecia Within each of the functional groups of protein synth- esis, transcription, energy, and genes of unknown func- tion, a higher proportion of genes were meagerly