Discovery of Porcine microRNAs in Multiple Tissues by a Solexa Deep Sequencing Approach Sheng-Song Xie., Xin-Yun Li., Teng Liu, Jian-Hua Cao, Qiang Zhong, Shu-Hong Zhao* Key Lab of Agricultural Animal Genetics, Breeding, and Reproduction of Ministry of Education & Key Laboratory of Swine Genetics and Breeding of Ministry of Agriculture, Huazhong Agricultural University, Wuhan, People’s Republic of China Abstract 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. In an effort to gain further identification of miRNAs in the pig, we have applied the Illumina Solexa sequencing technology to carry out an in-depth analysis of the miRNA transcriptome in a pool of equal amounts of RNA from 16 different porcine tissues. From this data set, we identified 437 conserved and 86 candidate novel miRNA/miRNA* in the pig, corresponding to 329 miRNA genes. Compared with all the reported porcine miRNAs, the result showed that 112 conserved and 61 candidate novel porcine miRNA were first reported in this study. Further analysis revealed extensive sequence variations (isomiRs) of porcine miRNAs, including terminal isomiRs at both the 59 and 39 ends and nucleotide variants. Read counts of individual porcine miRNA spanned from a few reads to approximately 405541 reads, confirming the different level of expression of porcine miRNAs. Subsequently, the tissue expression patterns of 8 miRNAs were characterized by Northern blotting. The results showed that miR-145, miR-423-5p, miR-320, miR-26a, and miR-191 are ubiquitously expressed in diverse tissues, while miR-92, miR-200a, and miR-375 were selectively enriched and expressed in special tissues. Meanwhile, the expression of 8 novel porcine-specific miRNAs was validated by stem-loop RT-PCR, and one of these was detected by Northern blotting. Using the porcine miRNA array designed according to our Solexa results, 123 miRNAs were detected expression in porcine liver tissues. A total of 58 miRNAs showed differential expression between the Tongcheng (a Chinese indigenous fatty breed) and Large White pig breeds (a lean type pig). Taken together, our results add new information to existing data on porcine miRNAs and should be useful for investigating the biological functions of miRNAs in pig and other species. Citation: Xie S-S, Li X-Y, Liu T, Cao J-H, Zhong Q, et al. (2011) Discovery of Porcine microRNAs in Multiple Tissues by a Solexa Deep Sequencing Approach. PLoS ONE 6(1): e16235. doi:10.1371/journal.pone.0016235 Editor: Carlo Gaetano, Istituto Dermopatico dell’Immacolata, Italy Received August 24, 2010; Accepted December 17, 2010; Published January 25, 2011 Copyright: 2011 Xie et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by a grant from the the National High Science and Technology Foundation of China ‘‘863’’ (2010AA10A104 and 2008AA101008), the NSFC-Guangdong Key Project (U0631005), National Key Basic Research and Development Program of China (2006CB102105), and the creative team project of the Chinese Ministry of Education (IRT0831). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: shzhao@mail.hzau.edu.cn . These authors contributed equally to this work. Introduction MicroRNAs (miRNAs) belong to a group of single-stranded noncoding RNAs that are 21–23 nucleotides (nt) in length [1]. The first characterized miRNA was lin-4 in 1993, but miRNAs were not recognized as a distinct class of biological regulators with conserved functions until the early 2000s [2,3,4]. These molecules typically interact with target mRNAs through base pairing, predominantly between bases 2–8 (the ‘‘seed’’ region) at the 59 end of the miRNA and complementary sites in the target 39 untranslated regions (39 UTR, ‘‘seed matches’’) [5]. These interactions result in translational repression and/or deadenyla- tion of target mRNA [6,7]. In animals, a few miRNAs can regulate gene expression at the post-transcriptional level by encoding target protein mRNAs through perfect complementary base pairing that leads to mRNA degradation. A typical example is miR-196, which can direct the cleavage of HOXB8 mRNA in mouse embryos [8]. miRNA biogenesis occurs through a multistep process that involves the activities of two Ribonuclease IIIs called Drosha and Dicer [9,10]. Many miRNAs are expressed in a tissue-specific and/or stage-specific manner [11]. A single miRNA can have one to several hundred target mRNAs, and it is estimated that 30% of all protein-coding genes in humans are regulated by miRNAs [5]. Increasing evidence suggests that miRNAs play important roles in cell differentiation, proliferation, growth, apoptosis, and immune response [12,13,14]. miRNAs regulate target mRNAs and act as rheostats that make fine-scale adjustments to protein output [15]. miRNAs are involved in the normal functioning of eukaryotic cells, and dysregulation of miRNAs is associated with disease [16]. Several miRNAs have links with certain types of cancer [17]. Extensive studies have indicated diverse patterns of miRNA regulation. Mature miRNAs target the 39 UTR of genes by complementary base pairing [18]. Furthermore, mature miRNAs can alter gene expression by binding to the coding regions as well as to the 59 UTR [19,20]. The down-regulation of target mRNAs by miRNAs has been widely observed. Recent experiments also show that miRNAs can up-regulate target mRNAs in some cases [21]. However, the biological functions of most miRNAs and their precise regulatory mechanisms remain PLoS ONE | www.plosone.org 1 January 2011 | Volume 6 | Issue 1 | e16235