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Identification and Characterization of MicroRNAs in Macaca fascicularis by EST Analysis.

ABSTRACT: MicroRNAs (miRNAs) are small noncoding RNAs which repress gene expression at the posttranscriptional level. In this study, an expressed sequence tag (EST)-based combined method was applied for the detection of miRNAs in Macaca fascicularis which is used as a model animal extensively in medical experiments, particularly those involved with neuroscience and disease. Initially, previously known miRNA sequences from metazoans were used to blast with the EST databases of Macaca fascicularis, and then a range of filtering criteria was conducted to remove some pseudo ones. At last a total of 8 novel conserved miRNAs were identified; their functions were further predicted and analyzed. Together, our study provides insight into miRNAs and their functions in Macaca fascicularis, indicating that the EST analysis is an efficient and affordable alternative approach for identifying novel miRNA candidates.

SUBMITTER: Yang H

PROVIDER: S-EPMC3398586 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Identification and Characterization of MicroRNAs in Macaca fascicularis by EST Analysis.

Yang Hao H Zhang Haiyang H Zhu Lin L Zhang Chenyu C Li Donghai D

Comparative and functional genomics 20120705

MicroRNAs (miRNAs) are small noncoding RNAs which repress gene expression at the posttranscriptional level. In this study, an expressed sequence tag (EST)-based combined method was applied for the detection of miRNAs in Macaca fascicularis which is used as a model animal extensively in medical experiments, particularly those involved with neuroscience and disease. Initially, previously known miRNA sequences from metazoans were used to blast with the EST databases of Macaca fascicularis, and then ...[more]

PMID: 22829752

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Project description:Background and aimEndogenous retroviruses (ERVs) found in all vertebrates, including non-human primates (NHPs), are known to be genetically inherited. Thus, recent studies have explored ERVs for human immunodeficiency virus vaccine development using human ERV (HERV) due to the hypervariability of exogenous retroviruses which cause conventional vaccines to be ineffective. HERV was also found to be able to induce an immune response in cancer patients. This study aimed to identify and molecularly characterize ERVs from Indonesian NHPs: Macaca fascicularis and Macaca nemestrina. Then, we described the phylogenetic relationship of these isolates with those of the simian ERVs (SERVs) characterized in other species and countries.Materials and methodsFirst, 5 mL of whole blood samples was taken from 131 long-tailed macaques and 58 pig-tailed macaques in captive breeding facilities at Bogor, Indonesia, for DNA extraction. Next, the DNA samples were screened using the SYBR Green real-time polymerase chain reaction (PCR) technique with specific primers for env (simian retroviruses [SRV]1-5 7585U19 and SRV1-5 7695L21). Positive SERV results were those with cycle threshold (CT) values < 24 (CT < 24) and melting temperature (TM) ranges of 80°C-82°C. Then, whole-genome nucleotide sequences from two pig-tailed macaques samples detected as positive SERV were generated using a nucleic acid sequencing technique which utilized the walking primer method. Subsequently, the sequences were analyzed using bioinformatics programs, such as 4Peaks, Clustal Omega, and BLAST (NCBI). Subsequently, a phylogenetic tree was constructed using the neighbor-joining method in MEGA X.ResultsSYBR Green real-time PCR amplification results indicated that SERV (Mn B1 and Mn B140910)-positive samples had CT values of 22.37-22.54 and TM of 82°C. Moreover, whole-genome sequences resulted in 7991 nucleotide sequences, comprising long terminal repeat, gag, pro, pol, and env genes identical between the sequenced samples. Furthermore, the phylogenetic tree results indicated that both samples from M. nemestrina had 99%-100% nucleotide identities to the Mn 92227 sample identified at the National Primate Center University of Washington (NaPRC UW) which was imported from Indonesia in 1998, confirmed as a novel SERV strain. The phylogenetic tree results also indicated that although SERV whole-genome nucleotide and env amino acid sequences were clustered with SRV-2 (identity values of 82% and 79%, respectively), they had a 99%-100% nucleotide identity to Mn 92227. Meanwhile, the gag, pro, and pol amino acids were clustered with SRV-1, SRV-3, SRV-4, SRV-5, SRV-8, and SERV/1997, with 82% and 88% identity values.ConclusionBased on the SYBR Green real-time PCR profiles generated, similarities with Mn 92227 were observed. Subsequent phylogenetic analysis confirmed that both samples (Mn B1 and Mn B140919) from pig-tailed macaques in the country of origin were novel SERV strains at NaPRC UW. Therefore, it could be used in biomedical research on ERVs.

Dataset Information

Identification and Characterization of MicroRNAs in Macaca fascicularis by EST Analysis.

Publications

Identification and Characterization of MicroRNAs in Macaca fascicularis by EST Analysis.

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