Isolation, Characterization And Expression Analysis Of Porcine MicroRNA

MicroRNAs(miRNAs) are a class of noncoding RNAs that negatively regulate gene expression at the posttranscriptional level.There is increasing evidence to suggest that miRNAs play important roles in development;however,few studies have focused on miRNAs in pig.In order to identify miRNAs expressed in the pig,we constructed two small RNA libraries.Subsequently,combination with bioinformafic analysis,hundreds of miRNAs were found,The main experimental methods and results are as follows:1.A library of small RNAs from porcine skeletal muscle was constructed,57 distinct miRNAs were identified,of which 39 have not been reported earlier in the pig.Of these, 2 miRNAs appear to be novel and pig-specific.Surprisingly,these 2 differ only in a single nucleotide.A part of their primary transcript was cloned and confirmed by sequencing analysis.Alignment of the 2 sequences using ClustalW showed that the precursor sequences were almost identical,but the flanking sequences were different,indicating that these 2 novel miRNAs may represent rapidly evolved miRNAs in the pig genome.2.The expression pattems of 8 miRNAs were characterized by real-time polymerase chain reaction of 8 pig tissue samples.The ssc-let-7e and ssc-miR-181b miRNAs were expressed in all analyzed tissues.The ssc-let-7c,ssc-miR-125b,ssc-miR-new1,and ssc-miR-new2 miRNAs were expressed in several analyzed tissues,while ssc-miR-122 and ssc-miR-206 were specifically expressed in the liver and muscle,respectively.3.To discover new miRNAs in the pig,another small RNA cDNA library was constructed using a porcine RNA pool from 16 different tissues,including heart,liver, spleen,lung,kidney,skeletal muscle,stomach,small intestine,inner fat,thymus, mesenteric lymph node,submandibular lymph node,testicle,33 day gestation whole embryo,endometrium,and placenta.These tissues collected from a 4-month-old Meishan boar,a 1-week-old Duroc gilt,a 33-d whole embryo of Landrace pig and a pregnant Yorkshire sow,respectively.Followed Solexa sequencing,we obtained 4821809 total reads.After filtering low quality reads,3’,5’ adaptor and reads number whose size shorter than 18 nucleotides(nt),we obtained 3388980 reads,which containing 399963 unique sequences ranged from 18-30nt.4.The solexa sequencing data was analyzed using local BLAST and miRDeep program.Porcine homologous miRNAs were also predicated by miRDeep using known miRNA as seed.Finally,we found 460 homologous and 67 novel miRNAs including the 65 porcine miRNAs in miRBase.There are 269,194,164 identified porcine miRNAs perfect match to human,mouse and rat corresponding miRNAs,respectively.We also found 19 miRNA-like candidated new type small RNAs in the size range of 27～30nt from deep sequencing data using miRDeep.Based on the homology search to RNAdb2.0 database 66 homologous candidated porcine piwiRNAs(piRNAs) were found.A number of SNPs in the miRNAs were also detected and need to be validated.Our results add to existing data on porcine miRNAs and are useful for investigating the biological functions of miRNAs in porcine tissues development.