posttranscriptional level in liver is different between lean and fatty pigs at the same body weight. miR-16 was shown to regulate liver cell line HepG2 apoptosis by targeting BCL2 gene [47], is also detected differential expression between the two breeds. Other differentially expressed miRNAs have no reports at present. Our result indicated that these differentially expressed miRNAs between different breeds may play important roles related to liver development, liver cell apoptosis and energy metabolism. These results also indicate our miRNA array is an appropriate tool for porcine miRNAs expression detection. Materials and Methods Tissue sample collection and RNA isolation The following tissues were collected and used to generate small RNA libraries: the heart, liver, longissimus dorsi muscle, and stomach from a one-week-old Duroc gilt; the spleen, lung, kidney, small intestine, inner fat, thymus, mesenteric lymph node, submandibular lymph node, and testicle from a four-month-old Meishan boar. The endometrium and placenta from a pregnant Yorkshire sow, and a 33-day-old whole embryo from a Landrace sow. Samples were immersed in liquid nitrogen immediately after collection and then stored at 280uC. Total RNA was isolated from porcine tissues using the Trizol reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions; the samples were then pooled. The quality of total RNA was checked on an Agilent 2100 Bioanalyzer system. RNA samples were stored at -80uC until further use. All research involving animals were conducted according to the regulation (No. 5 proclaim of the Standing Committee of Hubei People’s Congress) approved by the Standing Committee of Hubei People’s Congress, and the ethics committee of Huazhong Agricultural University, P. R. China. The approved permit number for this study is ‘‘HBAC20091138’’. Small RNA library construction and sequencing For small RNA library construction and deep sequencing, approximately 20 mg of pooled RNA was used. The sequencing was performed as follows: RNA was purified by polyacrylamide gel electrophoresis (PAGE) to enrich molecules in the range of 16–30 nt, and proprietary adapters were ligated to the 59 and 39 termini of the RNA. The samples were used as templates for cDNA synthesis. The cDNA was amplified using the appropriate PCR cycles to produce sequencing libraries that were subjected to Illumina Genome Analyzer’s proprietary sequencing-by-synthesis method. Sequencing was carried out at the Beijing Genomics Institute (BGI, China). Sources of sequences and software Before performing the sequence annotation, some necessary sequence data and software were retrieved from known websites. Preliminary sequence assemblies for pig chromosomes can be viewed in Pre Ensembl, and the assembled sequence was downloaded from ftp://ftp.sanger.ac.uk/pub/S_scrofa/assemblies/ PreEnsembl_Sscrofa7/(updated, 14th July 2008). The raw sequence data of pigs, which is in the form of traces at the NCBI Ensembl Trace Archive, was downloaded from ftp://ftp.ncbi.nih.gov/pub/ TraceDB/sus_scrofa/. The ncRNA data was downloaded from the Rfam 9.0 database (http://rfam.janelia.org/). Pig mRNAs were retrieved from the NCBI Nucleotide database (http://www.ncbi.nlm. nih.gov/) using the input ‘‘(pig mRNA)’’ AND ‘‘Sus scrofa [porgn:_txid9823].’’ Known miRNAs/miRNA hairpins were ob- tained from miRBase (Release15.0) (http://microrna.sanger.ac.uk/ sequences/). The miRDeep package was downloaded from http:// Discovery of Porcine MicroRNA by Solexa Sequencing www.mdc-berlin.de/rajewsky/miRDeep. In addition to Perl (avail- able at http://www.perl.com/), the Vienna package (available at http://www.tbi.univie.ac.at/RNA), Randfold application (http:// bioinformatics.psb.ugent.be/software/details/Randfold), and NCBI BLAST package (http://www.ncbi.nlm.nih.gov/BLAST/download. shtml) that required dependencies were downloaded from their respective sites. The piRNA data was retrieved from RNAdb (http:// jsm-research.imb.uq.edu.au/rnadb/default.aspx). Data analysis Individual sequence reads with the base quality scores were produced by Solexa. Low-quality reads were strictly removed from the raw reads. After trimming the 39 adaptor sequence and removing the 59 adaptor contaminants, all identical sequences of sizes ranging from 18–30 nt were counted and eliminated from the initial data set. The resulting unique sequences with associated read counts are referred to as sequence tags. All unique sequences were used to search the ncRNA data (Rfam) with BLASTN to remove non-miRNA sequences (rRNA, tRNA, etc.).Schematic illustration of the identification and character of porcine miRNAs was shown in Figure S1. Details of the three methods are as follows: Method 1, miRDeep program was used to identify porcine miRNAs from large pool of unique sequenced transcripts from our Solexa deep sequencing. All candidate Solexa RNA sequences between 18 nt and 30 nt were aligned against raw sequence data (NCBI Trace Archive) using the miRDeep software with the default parameter. Subsequently, the miRDeep miRNA precur- sors obtained from the short read library were searched against the pig genome (Ensembl Trace Archive) to determine the chromo- some location. Furthermore, putative novel miRNAs were searched against the NCBI NR database to determine whether these miRNAs exist in other species. These miRNA precursor sequences were then used for fold-back secondary structure prediction with the mfold program. Method 2, all unique candidate Solexa RNA sequences were re-queried against already known animal mature miRNAs (from the latest miRBase database, release 16.0) by using BLASTn software to the further identifica- tion of conserved porcine miRNAs. When the BLAST program was run, the parameter Expect value was set at 0.001 to improve the reliability. To identify more miRNAs in the pig genome, method 3 was re-adopted miRDeep program to predicate the porcine miRNAs, which using all known animal mature miRNAs (miRBase) instead of our Solexa reads aligned against raw pig genome sequence data. Finally, porcine miRNAs identified by the three above-described methods were combined. The putative origins of the remaining sequences were identified by BLASTN search against the pig mRNA data from NCBI. Sequences in the 26–30 nt size range, which were not annotated as miRNA, mRNA, rRNA, tRNA, etc., were used to search the piRNA data (RNAdb) with BLASTN and to check whether the small RNA library that we had generated contains piRNAs. Only perfect matches were classified as candidate piRNAs. Northern blotting Small RNA blot analysis was performed to determine the expression patterns of conserved and new miRNAs. Total RNA (20 mg) was resolved on a 15% denaturing polyacrylamide gel. RNA was electrophoretically transferred to HybondTM-N+ (Amersham) membranes, and the membranes were UV cross- linked and baked for 30 min at 80uC. DNA oligonucleotides complementary to small RNA sequences were end-labeled with c- 32P-ATP using T4 polynucleotide kinase (NEB), and these oligonucleotides were used as the probes (Table S17). The blots were pre-hybridized for at least 30 min and hybridized overnight PLoS ONE | www.plosone.org 9 January 2011 | Volume 6 | Issue 1 | e16235