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Identification of reference genes for relative quantification of circulating microRNAs in bovine serum.

Bae IS, Chung KY, Yi J, Kim TI, Choi HS, Cho YM, Choi I, Kim SH - PLoS ONE (2015)

Bottom Line: The results consistently showed that a combination of miR-93 and miR-127 provided the most stably expressed reference.Therefore, we conclude that this combination is the optimal endogenous reference for reverse transcription quantitative real-time polymerase chain reaction-based detection of microRNAs in bovine serum.The data presented in this study are crucial to successful biomarker discovery and validation for the diagnosis of physiopathological conditions in cattle.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Kyung Hee University, Seoul, Republic of Korea.

ABSTRACT
Circulating microRNAs in body fluids have been implicated as promising biomarkers for physiopathology disorders. Currently, the expression levels of circulating microRNAs are estimated by reverse transcription quantitative real-time polymerase chain reaction. Use of appropriate reference microRNAs for normalization is critical for accurate microRNA expression analysis. However, no study has systematically investigated reference genes for evaluating circulating microRNA expression in cattle. In this study, we describe the identification and characterization of appropriate reference microRNAs for use in the normalization of circulating microRNA levels in bovine serum. We evaluated the expression stability of ten candidate reference genes in bovine serum by using reverse transcription quantitative real-time polymerase chain reaction. Data were analyzed using geNorm, NormFinder, and BestKeeper statistical algorithms. The results consistently showed that a combination of miR-93 and miR-127 provided the most stably expressed reference. The suitability of these microRNAs was validated, and even when compared among different genders or breeds, the combination of miR-93 and miR-127 was ranked as the most stable microRNA reference. Therefore, we conclude that this combination is the optimal endogenous reference for reverse transcription quantitative real-time polymerase chain reaction-based detection of microRNAs in bovine serum. The data presented in this study are crucial to successful biomarker discovery and validation for the diagnosis of physiopathological conditions in cattle.

No MeSH data available.


Related in: MedlinePlus

Levels of candidate reference miRNAs among different genders.qRT-PCR experiments were performed on steer serum (steer; n = 14), bull serum (bull; n = 10), and heifer serum (heifer; n = 9). Boxes (blank for steer, diagonal lines for bull, and diamonds for heifer) represent lower and upper quartiles with median.
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pone.0122554.g005: Levels of candidate reference miRNAs among different genders.qRT-PCR experiments were performed on steer serum (steer; n = 14), bull serum (bull; n = 10), and heifer serum (heifer; n = 9). Boxes (blank for steer, diagonal lines for bull, and diamonds for heifer) represent lower and upper quartiles with median.

Mentions: To validate further the stability of miR-93, miR-127, and the combination of these two as reference miRNAs, we investigated their expression levels in bovine serum derived from different genders and breeds. First, the levels of candidate miRNAs were analyzed in steer, bull, and heifer serum. miR-93, miR-127, and miR-93/miR-127 were the most highly expressed in all genders, when compared to miR-192 and miR-101 (Fig 5). The Cq value ranges of miR-127 and miR-93/miR-127 were 1.36 and 1.57, respectively. Standard deviation within different gender groups for miR-93, miR-127, and miR-93/miR-127 was less than 1 (S2 Table). Between different gender groups, miR-92/miR-127 showed the least variation, with a p-value of 0.859, the highest value among all the candidate miRNAs. In addition, within different breeds (Holstein dairy cows and Korean native cattle), miR-127 and miR-93/miR-127 had standard deviations of less than 1 (Fig 6 and S3 Table). Only miR-93/miR-127 showed no statistical difference between breeds (p = 0.85). The Cq value range of miR-93/miR-127 in bovine serum extracted from different breeds was the narrowest among the candidate miRNAs. Based on data from different genders and breeds, the combination miR-93/miR-127 is recommended as the optimal reference for miRNAs in bovine serum.


Identification of reference genes for relative quantification of circulating microRNAs in bovine serum.

Bae IS, Chung KY, Yi J, Kim TI, Choi HS, Cho YM, Choi I, Kim SH - PLoS ONE (2015)

Levels of candidate reference miRNAs among different genders.qRT-PCR experiments were performed on steer serum (steer; n = 14), bull serum (bull; n = 10), and heifer serum (heifer; n = 9). Boxes (blank for steer, diagonal lines for bull, and diamonds for heifer) represent lower and upper quartiles with median.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4380332&req=5

pone.0122554.g005: Levels of candidate reference miRNAs among different genders.qRT-PCR experiments were performed on steer serum (steer; n = 14), bull serum (bull; n = 10), and heifer serum (heifer; n = 9). Boxes (blank for steer, diagonal lines for bull, and diamonds for heifer) represent lower and upper quartiles with median.
Mentions: To validate further the stability of miR-93, miR-127, and the combination of these two as reference miRNAs, we investigated their expression levels in bovine serum derived from different genders and breeds. First, the levels of candidate miRNAs were analyzed in steer, bull, and heifer serum. miR-93, miR-127, and miR-93/miR-127 were the most highly expressed in all genders, when compared to miR-192 and miR-101 (Fig 5). The Cq value ranges of miR-127 and miR-93/miR-127 were 1.36 and 1.57, respectively. Standard deviation within different gender groups for miR-93, miR-127, and miR-93/miR-127 was less than 1 (S2 Table). Between different gender groups, miR-92/miR-127 showed the least variation, with a p-value of 0.859, the highest value among all the candidate miRNAs. In addition, within different breeds (Holstein dairy cows and Korean native cattle), miR-127 and miR-93/miR-127 had standard deviations of less than 1 (Fig 6 and S3 Table). Only miR-93/miR-127 showed no statistical difference between breeds (p = 0.85). The Cq value range of miR-93/miR-127 in bovine serum extracted from different breeds was the narrowest among the candidate miRNAs. Based on data from different genders and breeds, the combination miR-93/miR-127 is recommended as the optimal reference for miRNAs in bovine serum.

Bottom Line: The results consistently showed that a combination of miR-93 and miR-127 provided the most stably expressed reference.Therefore, we conclude that this combination is the optimal endogenous reference for reverse transcription quantitative real-time polymerase chain reaction-based detection of microRNAs in bovine serum.The data presented in this study are crucial to successful biomarker discovery and validation for the diagnosis of physiopathological conditions in cattle.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Kyung Hee University, Seoul, Republic of Korea.

ABSTRACT
Circulating microRNAs in body fluids have been implicated as promising biomarkers for physiopathology disorders. Currently, the expression levels of circulating microRNAs are estimated by reverse transcription quantitative real-time polymerase chain reaction. Use of appropriate reference microRNAs for normalization is critical for accurate microRNA expression analysis. However, no study has systematically investigated reference genes for evaluating circulating microRNA expression in cattle. In this study, we describe the identification and characterization of appropriate reference microRNAs for use in the normalization of circulating microRNA levels in bovine serum. We evaluated the expression stability of ten candidate reference genes in bovine serum by using reverse transcription quantitative real-time polymerase chain reaction. Data were analyzed using geNorm, NormFinder, and BestKeeper statistical algorithms. The results consistently showed that a combination of miR-93 and miR-127 provided the most stably expressed reference. The suitability of these microRNAs was validated, and even when compared among different genders or breeds, the combination of miR-93 and miR-127 was ranked as the most stable microRNA reference. Therefore, we conclude that this combination is the optimal endogenous reference for reverse transcription quantitative real-time polymerase chain reaction-based detection of microRNAs in bovine serum. The data presented in this study are crucial to successful biomarker discovery and validation for the diagnosis of physiopathological conditions in cattle.

No MeSH data available.


Related in: MedlinePlus