Gunaratne et al. BMC Genomics 2011, 12:277 http://www.biomedcentral.com/1471-2164/12/277 pairs of pooled auditory forebrain samples. 1) Males (samples S7 and S8): 6 birds per pool, collected in November 2008. 2) Females (samples S1 and S2): 6 birds per pool, collected in May 2009. 3) Mixed (sam- ples S3 and S4), 3 males and 3 females each pool, col- lected in May 2009. Samples with odd numbers were from birds hearing song, and even number hearing silence. For Northern analysis: Auditory forebrains of 22 birds (12 females and 10 males) were collected in April 2009, and total RNA was extracted by Tri-Reagent (Ambion). Male and female samples were pooled after extraction. For TaqMan analysis: Analyses were performed on total RNA extracted either by mirVana or Tri-Reagent (Ambion), from the auditory forebrains of individual males or females, collected in April-August 2009, March 2010 or December 2010. Illumina small RNA sequencing and novel miRNA discovery Fifteen micrograms of total RNA from auditory fore- brain of song bird samples described above were gel- fractionated to isolate 18-30 nt small RNAs. 3’ and 5’ adapters were ligated to the small RNAs and constructs amplified following RT-PCR following the conditions specified in the small RNA kit (FC-102-1009, Illumina) protocol. The small RNA library was sequenced using a Solexa/Illumina GA-1 Genome analyzer. Small RNA sequences were analyzed through a high-throughput computational pipeline described by [28,29,90,91]. To identify zebra finch miRNAs that are also conserved in chicken, human and mouse, we performed a local Smith-Waterman alignment of each unique sequence read against each of the mature miRNAs in miRBase version 13.0 for each of these species. We allowed for a 3 base overhang on the 5’ end and a 6 base overhang on the 3’ end. In the case of redundantly aligning reads, mature miRNA sequences were equally apportioned among each of the hairpins. For each sample, all sequence reads were aligned to a reference set of pre- cursor miRNAs from miRBase version 13.0. The reads that did not align to any known miRNA were passed to our novel miRNA discovery platform as previously described [28]. Briefly, each sequence is first mapped to the reference genome sequence (WUGSC 3.2.4) and 200 bases of flanking sequence are extracted to further define the putative hairpin. This extracted sequence is then folded using the Vienna RNA folding package [92] and those sequences that form a plausible hairpin are selected as potential novel miRNA hairpins. These can- didates are filtered through a set of three Ambros cri- teria: 1) the mature putative miRNA sequence must rest on one side of a single hairpin; 2) the putative miRNA sequence must bind relatively tightly within the hairpin Page 10 of 14 stem containing no large or energetically unfavorable loops; and 3) the putative hairpin must have a miRNA- appropriate energy (free energy below -20 kcal/mol). All sequences that passed were then carefully curated to determine if Drosha and Dicer processing could yield the resulting mature sequence from the predicted hair- pin. These candidates are then divided into four differ- ent categories: “not likely”, “potential”, “high confidence”, and “confirmed” (as in red, gray, blue and green colors in Additional File 1, Tables S2 and S3). Candidates that are flagged red as “not likely” either failed to map in a pile of sequences in a very tight space of 15-25 nt of the predicted hairpin (e.g. were scattered evenly across the full length of the hairpin), mapped within the loop of the hairpin, or mapped to known tRNAs or rRNAs. Candidates that passed all of the above criteria, and also mapped within a hairpin with predicted Drosha and Dicer cut sites were categorized as “high confidence” (blue annotation in Additional File 1, Tables S2 and S3). All high confidence candidates for which we detected both the mature sequence and the putative star sequence from the same hairpin we cate- gorized as “confirmed” (green annotation in Additional File 1, Table S3). In addition to miRNA precursors, the reads were also mapped to the reference zebra finch genome using the Pash software package [93,94], and uploaded to the Genboree platform (http://www.gen- boree.com) to identify potential mappings to piRNAs, snoRNAs and other annotations in addition to miRNAs (data shown in Additional File 1, Table S1). PiRNAs (i. e., Piwi-interacting RNAs) have a central role in the maintenance of the integrity of genomes through the silencing of transposable elements [95]. SnoRNAs (small nucleolar RNAs) function in site-specific ribosomal RNA modification, rRNA processing and more recently have been found to guide alternate splicing and RNA editing of mRNA transcripts [96]. TaqMan qPCR To measure the mature miRNA, the TaqMan Micro- RNA Assay Kit (Applied Biosystems) was used accord- ing to the manufacturer’s instructions. Probe sequences used for each target miRNA are given in Table 4. Northern Blot Analysis Northern blotting to confirm novel miRNA tgu-miR- 2954-3p was performed by modifying the protocol of [97]. 2 μg of total RNA was heated at 65°C for 5 min with 2X loading dye (Ambion), quenched on ice, and loaded on a 15% TBE Urea gel (Invitrogen). Total RNA was separated by electrophoresis at 200V for 50 min. The gel was stained with with EtBr in 1x TBE (4 μL of 10 mg/ml EtBr per 100 ml of 1x TBE) for 3 minutes with gentle shaking and transferred to nylon membrane for 90