Identification of Reference Genes for Relative Quantification of Circulating MicroRNAs in Bovine Serum

Bae, In-Seon; Chung, Ki Yong; Yi, Jongmin; Kim, Tae Il; Choi, Hwa-Sik; Cho, Young-Moo; Choi, Inho; Kim, Sang Hoon

doi:10.1371/journal.pone.0122554

Cited by 22 publications

(11 citation statements)

References 35 publications

(47 reference statements)

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“…Recent studies show that the expression levels of RNU6A and some other small nuclear RNAs might be unstable in certain tissues and conditions and may not serve as best reference genes [ 26 ][ 27 ][ 28 ][ 29 ]. One recent publication has also identified miR-127-3p as a good candidate for reference gene selection [ 30 ].…”

Section: Methodsmentioning

confidence: 99%

Analysis of Deregulated microRNAs and Their Target Genes in Gastric Cancer

et al. 2015

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BackgroundMicroRNAs (miRNAs) are widely studied non-coding RNAs that modulate gene expression. MiRNAs are deregulated in different tumors including gastric cancer (GC) and have potential diagnostic and prognostic implications. The aim of our study was to determine miRNA profile in GC tissues, followed by evaluation of deregulated miRNAs in plasma of GC patients. Using available databases and bioinformatics methods we also aimed to evaluate potential target genes of confirmed differentially expressed miRNA and validate these findings in GC tissues.MethodsThe study included 51 GC patients and 51 controls. Initially, we screened miRNA expression profile in 13 tissue samples of GC and 12 normal gastric tissues with TaqMan low density array (TLDA). In the second stage, differentially expressed miRNAs were validated in a replication cohort using qRT-PCR in tissue and plasma samples. Subsequently, we analyzed potential target genes of deregulated miRNAs using bioinformatics approach, determined their expression in GC tissues and performed correlation analysis with targeting miRNAs.ResultsProfiling with TLDA revealed 15 deregulated miRNAs in GC tissues compared to normal gastric mucosa. Replication analysis confirmed that miR-148a-3p, miR-204-5p, miR-223-3p and miR-375 were consistently deregulated in GC tissues. Analysis of GC patients’ plasma samples showed significant down-regulation of miR-148a-3p, miR-375 and up-regulation of miR-223-3p compared to healthy subjects. Further, using bioinformatic tools we identified targets of replicated miRNAs and performed disease-associated gene enrichment analysis. Ultimately, we evaluated potential target gene BCL2 and DNMT3B expression by qRT-PCR in GC tissue, which correlated with targeting miRNA expression.ConclusionsOur study revealed miRNA profile in GC tissues and showed that miR-148a-3p, miR-223-3p and miR-375 are deregulated in GC plasma samples, but these circulating miRNAs showed relatively weak diagnostic performance as sole biomarkers. Target gene analysis demonstrated that BCL2 and DNMT3B expression in GC tissue correlated with their targeting miRNA expression.

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Section: Methodsmentioning

confidence: 99%

Analysis of Deregulated microRNAs and Their Target Genes in Gastric Cancer

et al. 2015

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“…Relative quantification was performed by normalization of the expression of target miRNAs against that of miR-191, used as endogenous control. miR-191 has previously been reported to have stable expression in bovine serum [ 30 ] and in bovine oocytes and early embryos, and porcine oocytes [ 31 ] and thus is suitable as optimal endogenous expression normalizer for reverse transcription quantitative real-time polymerase chain reaction-based detection of microRNAs.…”

Section: Resultsmentioning

confidence: 99%

Identification of the MicroRNA Repertoire in TLR-Ligand Challenged Bubaline PBMCs as a Model of Bacterial and Viral Infection

et al. 2016

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In the present study, we used high-throughput sequencing, miRNA-seq, to discover and explore the expression profiles of known and novel miRNAs in TLR ligand-stimulated vis-à-vis non-stimulated (i.e. Control) peripheral blood mononuclear cells (PBMCs) isolated from blood of healthy Murrah buffaloes. Six small RNA (sRNA) libraries were multiplexed in Ion Torrent PI chip and sequenced on Ion Proton System. The reads obtained were aligned to the Bos taurus genome (UMD3.1 assembly), which is phylogenetically closest species to buffalo (Bubalus bubalis). A total of 160 bovine miRNAs were biocomputationally identified in buffalo PBMCs and 130 putatively novel miRNAs (not enlisted in the bovine mirBase) were identified. All of these 290 miRNAs identified across the six treatment and control samples represent the repertoire of novel miRNAs for the buffalo species. The expression profiles of these miRNAs across the samples have been represented by sample dendrogram and heatmap plots. The uniquely expressed miRNAs in each treatment and control groups were identified. A few miRNAs were expressed at very high levels while the majority of them were moderately expressed. The miRNAs bta-miR-103 and -191 were found to be highly abundant and expressed in all the samples. Other abundantly expressed miRNAs include bta-miR-19b, -29b, -15a, -19a, -30d, -30b-5p and members of let family (let 7a-5p, let 7g & let 7f) in LPS and CpG treated PBMCS and bta-miR-191, -103 & -19b in Poly I:C stimulated PBMCs. Only one novel miRNA (bta-miR-11039) out of 130 identified putatively novel miRNAs, was expressed in all the six samples and differentially expressed (>2- fold) miRNAs were identified. Six of the differentially expressed miRNAs across the groups (bta-miR-421, bta-let-7i, bta-miR-138, bta-miR-21-5p, bta-miR-222 and bta-miR-27b) were subsequently confirmed by TaqMan quantitative reverse transcription polymerase chain reaction (qRT-PCR). Furthermore, the target genes of differentially expressed miRNAs were enriched for the roles in innate immunity and TLR signaling pathways. This maiden study on profiling and cataloguing of bubaline miRNAs expressed in TLR-ligand stimulated PBMCs will provide an important reference point for future studies on regulatory roles of miRNAs in immune system of buffaloes.

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“…In this study, miR-93 was used as an internal reference to calculate the relative expression of target miRNA using the 2 -ΔΔCT (cycle threshold, CT) method [ 61 ]. This miRNA was selected due to its stable expression between sera and exosomes after comparing with the most used reference U6 [ 62 , 63 ], and previously reported serum miRNA reference candidates miR-127 and miR-744 in bovine and mouse [ 64 , 65 ]. In addition, no difference was observed between sera and exosomes in miR-93 expression detected by RNA-seq ( P > 0.05).…”

Section: Methodsmentioning

confidence: 99%

Comparative miRNAome analysis revealed different miRNA expression profiles in bovine sera and exosomes

et al. 2016

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BackgroundExtra-cellular components, such as serum and exosome, have drawn great attention as a readily accessible source of biomarkers for mammalian health. However, the contribution of different blood components to the signature of respective microRNAs (miRNAs) remains unknown, especially in cattle. In this study we aimed to investigate the miRNAs from bovine sera and exosomes, and to provide insights into their future applications.ResultsBlood collected from four healthy dairy cows were used for this study. The serum and exosomal RNAs were extracted using two commonly used commercial kits (Norgen and Invitrogen total RNA isolation kit), respectively. The miRNA profiles were then generated using RNA-seq. Sera had higher complexity of miRNAome consisting of 328 ± 17 miRNAs, while less number of miRNAs (260 ± 15, P = 0.001) was detected in exosomes. The profile of total detected miRNAs in sera and exosomes was different, while exosomes represented about 78 % of total miRNAs expressed in sera, suggesting that exosomes are the major miRNAs carriers in bovine sera. A total of 24 and 3 miRNAs (RPM > 5) were exclusively expressed in sera and exosomes, respectively. In addition, 12 miRNAs were differentially expressed between sera and exosomes (FDR < 0.05), with the expression of four of them being further validated by stem-loop RT-qPCR. Moreover, functional analysis showed that uniquely and highly expressed miRNAs in sera were mainly related to diseases and disorders, while the predicted functions of those in exosomes were enriched in tissue development and lipid metabolism.ConclusionOur results provide evidence that bovine sera and exosomes miRNAomes are different with regarding to the miRNA numbers, types and expressions. Based on their distinct profiles, miRNAs from sera and exosomes may reflect different aspects of physiological and pathological conditions in cattle. The functional analysis suggest that sera may be preferable for the purpose of detecting inflammation in cattle, while exosomes may be a better choice for monitoring the status of muscle development and lipid metabolism.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2962-1) contains supplementary material, which is available to authorized users.

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Identification of Reference Genes for Relative Quantification of Circulating MicroRNAs in Bovine Serum

Cited by 22 publications

References 35 publications

Analysis of Deregulated microRNAs and Their Target Genes in Gastric Cancer

Analysis of Deregulated microRNAs and Their Target Genes in Gastric Cancer

Identification of the MicroRNA Repertoire in TLR-Ligand Challenged Bubaline PBMCs as a Model of Bacterial and Viral Infection

Comparative miRNAome analysis revealed different miRNA expression profiles in bovine sera and exosomes

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