201 porcine homolog (identity.95%) miRNAs were detected (Table S7). The corresponding porcine miRNA precursor sequences, secondary structure and genome location were listed in Table S8. Some known porcine miRNAs were also successfully detected by miRDeep, such as ssc-let-7c, ssc-miR-103, and ssc- miR-122. The results indicated that miRDeep can be used not only to analyze high-throughput sequence data but also to predict miRNAs from genomic sequences. Sequence variations in miRNAs In our Solexa library, porcine miRNAs frequently exhibited variation from their ‘‘reference’’ sequences, producing multiple mature variants that we hereafter refer to as isomiRs. The miRNA* sequence and its isomiRs were also observed in this library. Evidence of isomiRs of a similar nature were detected in a limited sequence analysis using a linker based miRNA cloning approach in our previous studies from a single RNA pool [30], suggesting that isomiRs are not due to artifacts created from massively parallel sequencing (Figure. 3). All isomiRs are listed in Table S10. In total, we found 23822 isomiRs for a total of 459 sequenced miRNAs and miRNA*s (Table S10). The kinds of different isomiRs for a given miRNA range from one to 1051(ssc- miR-22a and ssc-let-7b) and 56 miRNAs exhibit more than one hundred kinds of isomiRs. There are 64 miRNAs that have no isomiRs. In 77 cases, this most abundant sequence did not correspond exactly to the current porcine miRBase 16.0 reference sequence (Table S10). For example, we found that the size of ssc- miR-29b in our library is 23 (read number is 88), while the size of miR-29b in miRBase is 20 (read number is 11). The size of another miRNA ssc-miR-505 in our library is 18 (read number is 6), while the size of miR-505 in miRBase is 20 (read number is only 1). It seems that under the different physiological conditions, the process length of miRNAs may vary, which produce different isomiRs. This also suggests either that the relative abundance of isomiRs may vary across different studies or that the original submission of this miRNA to miRBase was incorrect. Following the most recent update to miRBase, far more of the most abundant sequences in our library corresponded with the updated miRBase reference, pointing toward the latter explanation. Determination of tissue expression patterns of precursor and mature miRNAs by Northern blotting miRNA expression patterns are generally examined to obtain a better understanding of the physiological functions of these molecules. Moreover, in vivo expression analysis provides addi- tional evidence for the existence of bona fide miRNAs. To experimentally confirm and validate the results obtained from the Solexa library, we examined the expression patterns of conserved miRNAs. These miRNAs were randomly selected and experimentally verified by Northern blotting hybridization in 10 normal pig tissues, i.e., the heart, liver, spleen, lung, kidney, skeletal muscle, stomach, small intestine, lymph node, and brain. Antisense RNA probes that were designed to hybridize to mature miRNA and 5S rRNA are listed in Table S17. Using these probes, miR-145, miR-192, miR-320, and miR-423-5p showed signals that were approximately 22 nt in size, while the other probes detected not only the mature form but also the approximately 75- nt precursors on the same blots (Figure 4). The results also showed that the tissue expression patterns or the expression levels of the precursor and mature forms differed. For example, the precursor forms of mir-375 and mir-200a were ubiquitously expressed in 10 tissues, while mature miR-375 was only detected in the stomach and lymph node. Moreover, miR-200a was undetectable in the heart, spleen, skeletal muscle, and brain. Although the expression Discovery of Porcine MicroRNA by Solexa Sequencing patterns of the precursor and mature forms of miR-26a and miR- 191 were consistent across tissues, the expression levels of the precursor forms of mir-26a and mir-191 were much lower than those of their mature forms. The expression patterns of some miRNAs indicated selective expression in some tissues, for example, miR-192 was only expressed in the kidney, stomach, and small intestine at low levels. miR-200a was highly expressed in the kidney, lymph node, and stomach and was moderately expressed in the other tissues. miR-320 was abundantly expressed in the lungs but was expressed in trace amounts in other tissues. miR-145 was expressed at high levels in the spleen, lung, and small intestine, and miR-26a was relatively strongly expressed in the heart, lung, and kidney (Figure 4). Stem-loop RT-qPCR The stem-loop RT-qPCR assay was used to specifically detect mature miRNAs, as reported previously [39]. To validate novel candidate miRNAs found by Solexa sequence and miRDeep approach, thirty-five miRNAs were randomly selected to perform end-point RT-PCR (Table S17). The results showed that PCR products of the predicted size could be obtained using template cDNA, and the control without the template (water) gave no signals (Figure 5). Using this approach, 8 candidate novel miRNAs (ssc-miR-large1, ssc-miR-large13, ssc-miR-large16, ssc-miR- large19, ssc-miR-new47, ssc-miR-new49, ssc-miR-new54, and ssc-miR-new62) could be amplified and their specific bands could be detected by stem-loop RT-PCR (Figure 5, A). No specific band was detected for the other selected miRNAs. Among these, only one miRNA, i.e., ssc-miR-new62, was detected and expressed in the skeletal muscle and spleen and in trace amounts in other tissues, as determined by Northern blotting. The results confirmed that this novel miRNA exists in pigs (Figure 5, B). The signals of several candidate novel miRNAs were not detected by the above Northern blotting method, and it is possible that these may have relatively low expression levels in the selected tissues. Detection of differential expression in liver between fatty and lean pigs using a self-designed mammalian miRNA microarray All porcine miRNAs which obtained using the above methods were listed in Table S12. Subsequently, we designed a miRNAs array using all porcine miRNAs that are listed in this Table and miRNAs of other species in miRBase. Using this porcine miRNA array, 123 miRNAs were detected expression in porcine liver tissues (with a positive rate 22.5%), which including 19 new miRNAs detected by Solexa deep sequencing. The top 10 miRNAs which highly expressed in the liver were miR-720, miR-122, miR-923, miR-1826, miR-1260, miR-large1, miR- new51, miR-new34, mirR-894 and mirR-new36. Four among them were new miRNAs which detected by Solexa deep sequencing. According to our microarray result, we found there were 58 miRNAs differentially expressed (LogFC$|61.5|, P,0.05) between the Tongcheng and Large White pig breeds (Table S13). In order to verification the differential expressed miRNAs, four down-regulated (let-7a, miR-16, miR-20a, miR- 20b-5p) and 4 up-regulated miRNAs (miR-143, miR-192, miR- 133b, miR-223) were randomly selected and analyzed using Real- time PCR method (Figure 6, A). Then, the Pearson correlation coefficient of the Real-time PCR assay and the microarray assay was conducted and the value in down-regulated miRNA dateset is 0.7955, in up-regulated miRNAs dateset is 0.8889 (Figure 6, B). This result indicated that there was good correlation between the microarray analysis and the qPCR analysis. These results PLoS ONE | www.plosone.org 4 January 2011 | Volume 6 | Issue 1 | e16235