Gunaratne et al. BMC Genomics 2011, 12:277 http://www.biomedcentral.com/1471-2164/12/277 human but not chicken assemblies). Tgu-mir-2976 maps to three loci in the finch and 14 in the chicken, indicat- ing a probable expansion of this miRNA in the chicken lineage. This putative novel miRNA is not currently detected in the human assembly HG18. Tgu-mir-2985 is intriguing as it is located within two stem loops within the introns of two functionally related genes: the gluta- mate receptor subunits GRIA2 and GRIA4 in all three genomes. miRNA responses to song exposure When zebra finches are exposed to playback of a song they have not heard recently, changes occur in the expression of many different mRNAs as detected 30 min after stimulus onset [14]. To determine whether specific miRNAs also change in expression, we counted the Illumina reads in samples of RNA pooled from the auditory forebrain of birds either 30 min after onset of song playback (Song group) or from matched controls (Silence group). In our first such experiment, the birds in both groups were all males (n = 6 each). The read count for each miRNA in each sample was normalized to the total number of usable reads mapped in that sam- ple. We then calculated the ratio of the normalized count in the Song-stimulated condition compared to the Silence condition and performed a Fisher’s exact test (with correction for multiple testing) to evaluate whether the ratio differed significantly from the range of expected values at a 95% confidence interval. In the initial experiment with males, 49 of the known con- served miRNAs showed a significant difference, with 28 decreasing and 21 increasing in the group exposed to song (Additional File 1, Table S4). To address the biological reproducibility of the miRNA responses to song more broadly, we then repeated the small RNA-seq comparison two additional times using new groups of birds. In the second experiment, we used only females, and in the third we used an equal mix of Page 4 of 14 males and females. In total, therefore, we performed three independent “song-silence” pairwise comparisons by small RNA-seq, with an overall sex balance but differ- ent sex ratios in each individual comparison. These sec- ond and third experiments were done six months after the first and Illumina technology had improved by this time so that we obtained twice as many read counts (Table 1) - but again we normalized to the total mapped read number in each individual sample for our statistic analyses. As in the first experiment, we again observed differential read counts for roughly a third of the miR- NAs, but the identities of the miRNAs affected were somewhat different in each comparison. This is summar- ized graphically as a Venn diagram (Additional File 2, Figure S1), and comprehensive read count data are pre- sented in Additional File 1, Table S4. Across all three experiments, five conserved miRNAs showed changes that were both significant and in same direction in all comparisons (Table 2). For a number of other miRNAs, including let-7f, an apparent effect of song exposure was measured in all three experiments but the direction of change was not consistent (Additional File 1, Table S4). We performed TaqMan assays on RNA from additional birds, probing for eleven of the “significantly affected” miRNAs, and obtained fluorescent signals in PCR for ten. In nine out of ten cases, we observed the same direction of song response by TaqMan as in the small RNA-seq experiment, although the P-value by TaqMan was below 0.05 in only five cases (tgu-miR-124, tgu- miR-29a, tgu-miR-92, tgu-129-5p, and tgu-miR-2954-3p, Additional File 1, Table S4). The lack of statistical sig- nificance in the TaqMan assay for the others could reflect differences in the sensitivity and resolution of Illumina vs. TaqMan assays, or the operation of other uncontrolled factors in our experiments that lead to variability in the expression of some miRNAs. The transcriptional response in the auditory forebrain of zenk and other mRNAs is specific to song relative to Table 2 Conserved miRNAs with consistent responses to song exposure Silence Song Fold Change FDR-P Silence Song Fold Change FDR-P Silence Song Fold Change FDR-P Increasing tgu-miR-25 tgu-miR-192 Decreasing tgu-miR-92 tgu-miR-124 tgu-miR-129-5p 227 26 359 24624 2020 423 69 100 7056 602 Male 3.57 5.08 0.53 0.55 0.57 1.6E-27 1.2E-06 1.1E-04 2.1E-251 4.0E-19 55 36 5479 56802 9778 212 90 5398 46434 7272 Female 3.60 2.33 0.92 0.76 0.69 1.4E-10 5.5E-03 5.5E-03 1.1E-206 2.8E-62 35 11 7461 50955 12128 160 97 6887 77220 9284 Mix 2.92 5.63 0.59 0.97 0.49 2.1E-05 4.3E-06 6.3E-108 1.6E-04 2.6E-293 Shown are the Illumina read data for the five miRNAs that show a consistent response to song (same direction of change, significant in all three comparisons). “Song” and “Silence” list raw counts from the Illumina read analysis (Additional File 1, Table S4). “Fold Change” is the ratio of Song versus Silence read counts, after the raw counts were normalized within each run to the sum of mapped reads for that sample. Thus a value of > 1 indicates a relative increase in the group exposed to song, and < 1 indicates a decrease. “FDR-P” indicates the result of the Fisher’s exact test (FDR adjusted) for this comparison. See Additional File 1, Table S4 for full list of values for all miRNAs, and associated TaqMan values for a subset of these miRNAs (measured in a different set of males and females).