Figure 4. Expression profiles of conserved porcine miRNAs by Northern Blotting. Total RNA isolation from porcine heart, liver, spleen, lung, kidney, skeletal muscle, stomach, small intestine, lymph node and brain, 5S rRNA served as a loading control. Hybridization signals were detected for pre-miRNAs or/and mature miRNAs. doi:10.1371/journal.pone.0016235.g004 Figure 5. Validation of candidate novel porcine miRNAs. A. Validation of novel miRNAs by Stem-loop RT-PCR; B. The expression profile of ssc-miR-62 was detected by Northern blotting. Total RNA isolation from porcine heart, liver, spleen, lung, kidney, skeletal muscle, stomach, small intestine, lymph node and brain, 5S rRNA served as a loading control. Hybridization signals were detected for mature new miRNA. doi:10.1371/journal.pone.0016235.g005 Discovery of Porcine MicroRNA by Solexa Sequencing indicated that our self-designed mammalian miRNA microarray was suitable for porcine miRNAs analysis. Moreover, these differentially expressed miRNAs may play important roles in the liver between fat and lean type pig breeds. Discussion The domestic pig (Sus scrofa) is an important economic animal for meat production and as a suitable model organism for comparative genomics and biomedical studies [22,23]. To identify more pig miRNAs, especially pig-specific novel miRNAs, a small RNA cDNA library from a mixture of different porcine tissues was constructed for deep sequencing using the Solexa sequencing technique. Subsequently, we used a combination of the miRDeep algorithm and the homology search method to obtain 437 conserved miRNAs and 86 candidate novel pig-specific miRNAs. All porcine miRNAs which obtained by using different methods were listed in Table S12. The genomic locations of the conserved miRNAs on the WGS sequence from the NCBI database were generated by method 1 and method 3 was listed in Table S8. Then, the chromosome location on the pig draft genomic sequence from the Sanger database was determined using the BLASTN software (Table S9). Among these, 188 conserved and 86 candidate novel miRNAs were found from the Solexa sequencing data by method 1, and 416 miRNAs were identified from the Solexa deep sequencing data using the method 2. Moreover, 137 miRNAs were predicted in the pig genome using the method 3 (Figure 7). As shown in Figure 7, 184, 184, and 137 miRNAs overlapped across the three different methods, respec- tively, and 135 miRNAs were simultaneously identified by the three methods. Comparison of the results of porcine miRNA sequence identification by different approaches revealed that some of the miRNA sequences have minor differences, which could be detected by using different methods. Due to the close phylogenetic distance between the pig and human, the majority of the sequence reads corresponded to previously annotated humans miRNAs, and only a few potential novel pig-specific miRNAs were obtained. In this study, we used direct sequencing and computer prediction methods to identify porcine miRNAs. However, we found that each method has certain limitations. The miRDeep software is designed for miRNA annotation from the new high- throughput sequencing data generated by Solexa, 454, etc. This program first maps the sequences to the genome and then indicates whether a sequence is an miRNA based on the structural features of Dicer enzyme processing and the frequencies of sequence reads [37]. In method 1, the main advantage of using miRDeep software is that it can simultaneously identify conserved and new miRNAs, and the accuracy is good because this software uses a probabilistic model of miRNA biogenesis to score the compatibility of the position and the frequency of the sequenced RNA with the secondary structure of the miRNA precursor. One disadvantage is that some new miRNAs identified by miRDeep are actually false positives. Thus, new miRNAs should be double checked with ‘‘mfold’’ software to confirm their identity. Another problem is that the complete genomic sequence of pigs is not available, and many miRNAs that do not align to the pig genome cannot be identified by miRDeep. Therefore, in addition to miRDeep, we used BLASTn program (method 2) to identify pig miRNAs. In general, miRNAs are conserved among species, and homology search can help in detecting miRNAs by comparing the Solexa sequences to miRNAs in miRBase even when the complete genomic sequence of the species is not known. However, the drawback of this method is that some miRNAs match the same miRNA in different species, and there are one or two base pair PLoS ONE | www.plosone.org 6 January 2011 | Volume 6 | Issue 1 | e16235