elusive. Therefore, much effort has been made to elucidate miRNA functions in recent years. Pigs are essential organisms in the study of human health because their physiology is similar to that of humans, and they have comparable organ size and body mass. These animals are used as models for studying the consequences of infectious disease, testing drugs, practicing new surgical techniques, performing xenotransplants, and exploring lifestyle diseases such as cardio- vascular disease and obesity [22,23]. Despite increasing evidence for the diverse roles of miRNAs in biological processes, obtained through homology search or/and small RNA cDNA library cloning, the progress of miRNA studies in pigs has been slow [24,25,26,27,28,29,30,31]. The number of porcine miRNAs explored has increased in recent times due to the application of new deep sequencing techniques such as those from 454 Life Sciences and Solexa, which are suitable for the sequencing of miRNAs. This has greatly accelerated porcine miRNA discovery [32,33,34]. However, identification of miRNAs in pigs has been limited to certain tissues such as the skeletal muscle, adipose, heart, liver, thymus, and intestinal tissues. We hypothesize that more miRNAs are present. In particular, pig-specific novel miRNAs may exist. To test our hypothesis, we constructed a small RNA cDNA library using pooled RNA from 16 porcine tissues, for example, heart, liver, spleen, lung, kidney, and skeletal muscle. These tissues were collected from the Meishan boar, Duroc gilt, Landrace pig, and Yorkshire sow. miRNAs were identified by Solexa sequencing followed by computer analysis. Hundreds of conserved and candidate novel miRNAs were identified. Our experiments have led to an increase in the number of porcine miRNAs identified. We also showed that almost all miRNAs exhibited isoforms of variable length and thus have potentially distinct function. Further more, a porcine miRNAs array was designed according to our Solexa sequencing results and the miRNAs in liver tissue of different pig breeds was analyzed using this array. Some differentially expressed miRNAs between different pig breeds were detected. These results should be particularly useful for studying the biological functions of miRNAs in pigs. Discovery of Porcine MicroRNA by Solexa Sequencing Results Overview of the Solexa sequencing data To increase the coverage of porcine miRNAs by Solexa sequencing, a small RNA library was constructed from the pooled porcine RNA samples collected from 16 tissues of young and adult pigs. Schematic illustration of the identification and character of porcine miRNAs was shown in Figure S1. The 3 different methods are described in detail in ‘‘material and methods’’ section. After Solexa sequencing, 4,821,809 raw reads were obtained from the small RNA library. The low-quality reads were removed, and the 39 adaptor sequence was trimmed. The 59 adaptor contaminants were also removed. Small RNA sequences ranging in size from 18–30 nt were retrieved from the raw data set. In total, we obtained 3,389,218 high-quality reads, which accounted for 70.30% of the total raw reads. To simplify the sequencing data, all identical sequence reads in the small RNA library were grouped and converted into sequence tags. Finally, we obtained 399,963 unique sequences with associated counts of individual sequence reads. The size distribution of the reads is shown in Figure 1. The majority of the small RNAs were 21–23 nt in size. Sequences that were 22 nt in length accounted for 43.69% of total sequence reads, which is the typical size range for Dicer-derived products. The 21- nt size class was also dominant. The composition of the RNA classes is shown in Figure 2 (Detail see Table S14). The small class of cloned RNAs represents fragments of less abundant noncoding RNAs (rRNA, tRNA, small nuclear RNA, small nucleolar RNA, and piRNA), and this was identified by BLASTN searches against the Rfam and RNAdb databases [35]. A fraction also represents mRNA breakdown products from the pig. The remaining sequences formed the annotated porcine miRNAs, and these accounted for 65.12% of the total sequence reads in this small RNA library (Figure 2, A). These results indicated that the small RNA library was highly enriched in miRNA sequences. However, the sequence reads that were derived from annotated miRNAs represented only a relatively small fraction (5.64%) of the total number of unique sequence tags (Figure 2, B). The highest fraction Figure 1. Sequence length distribution. doi:10.1371/journal.pone.0016235.g001 PLoS ONE | www.plosone.org 2 January 2011 | Volume 6 | Issue 1 | e16235