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Evaluation of reference gene suitability for quantitative expression analysis by quantitative polymerase chain reaction in the mandibular condyle of sheep.

Jiang X, Xue Y, Zhou H, Li S, Zhang Z, Hou R, Ding Y, Hu K - Mol Med Rep (2015)

Bottom Line: Reference genes are commonly used as a reliable approach to normalize the results of quantitative polymerase chain reaction (qPCR), and to reduce errors in the relative quantification of gene expression.The results obtained from the four methods demonstrated a similar trend: RPL19, ACTB, and PGK1 were the most stably expressed reference genes in the sheep mandibular condyle.As determined by RefFinder comprehensive analysis, the results of the present study suggested that RPL19 is the most suitable reference gene for studies associated with the sheep mandibular condyle.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Military Stomatology, Department of Oral and Maxillofacial Surgery, School of Stomatology, The Fourth Military Medical University, Xi'an, Shaanxi 710032, P.R. China.

ABSTRACT
Reference genes are commonly used as a reliable approach to normalize the results of quantitative polymerase chain reaction (qPCR), and to reduce errors in the relative quantification of gene expression. Suitable reference genes belonging to numerous functional classes have been identified for various types of species and tissue. However, little is currently known regarding the most suitable reference genes for bone, specifically for the sheep mandibular condyle. Sheep are important for the study of human bone diseases, particularly for temporomandibular diseases. The present study aimed to identify a set of reference genes suitable for the normalization of qPCR data from the mandibular condyle of sheep. A total of 12 reference genes belonging to various functional classes were selected, and the expression stability of the reference genes was determined in both the normal and fractured area of the sheep mandibular condyle. RefFinder, which integrates the following currently available computational algorithms: geNorm, NormFinder, BestKeeper, and the comparative ΔCt method, was used to compare and rank the candidate reference genes. The results obtained from the four methods demonstrated a similar trend: RPL19, ACTB, and PGK1 were the most stably expressed reference genes in the sheep mandibular condyle. As determined by RefFinder comprehensive analysis, the results of the present study suggested that RPL19 is the most suitable reference gene for studies associated with the sheep mandibular condyle. In addition, ACTB and PGK1 may be considered suitable alternatives.

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Candidate reference genes of the control group ranked using five different methods. (Y-axis, stability values of the reference genes).
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f6-mmr-12-04-5633: Candidate reference genes of the control group ranked using five different methods. (Y-axis, stability values of the reference genes).

Mentions: The combined results obtained from geNorm, BestKeeper, the ΔCt method, and NormFinder were analyzed comprehensively using RefFinder (Table VI and Fig. 5). In the control group, RefFinder identified ACTB as the most stably expressed gene, with a stability value of 1.00. In the test group, PGK1 was the most stable reference gene with a stability value of 2.06. The combined results of the two groups (combined group) identified RPL19 as the most stably expressed reference gene with a stability value of 1.32, whereas YWAZH was the least stable reference gene. Candidate reference genes of the control group, test group and combined group, ranked using five different methods, were compared in Figs. 6–8, respectively. Though each algorithm employs a different method to determine the most suitable reference gene, the results obtained from all of the methods exhibited a similar trend.


Evaluation of reference gene suitability for quantitative expression analysis by quantitative polymerase chain reaction in the mandibular condyle of sheep.

Jiang X, Xue Y, Zhou H, Li S, Zhang Z, Hou R, Ding Y, Hu K - Mol Med Rep (2015)

Candidate reference genes of the control group ranked using five different methods. (Y-axis, stability values of the reference genes).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4581798&req=5

f6-mmr-12-04-5633: Candidate reference genes of the control group ranked using five different methods. (Y-axis, stability values of the reference genes).
Mentions: The combined results obtained from geNorm, BestKeeper, the ΔCt method, and NormFinder were analyzed comprehensively using RefFinder (Table VI and Fig. 5). In the control group, RefFinder identified ACTB as the most stably expressed gene, with a stability value of 1.00. In the test group, PGK1 was the most stable reference gene with a stability value of 2.06. The combined results of the two groups (combined group) identified RPL19 as the most stably expressed reference gene with a stability value of 1.32, whereas YWAZH was the least stable reference gene. Candidate reference genes of the control group, test group and combined group, ranked using five different methods, were compared in Figs. 6–8, respectively. Though each algorithm employs a different method to determine the most suitable reference gene, the results obtained from all of the methods exhibited a similar trend.

Bottom Line: Reference genes are commonly used as a reliable approach to normalize the results of quantitative polymerase chain reaction (qPCR), and to reduce errors in the relative quantification of gene expression.The results obtained from the four methods demonstrated a similar trend: RPL19, ACTB, and PGK1 were the most stably expressed reference genes in the sheep mandibular condyle.As determined by RefFinder comprehensive analysis, the results of the present study suggested that RPL19 is the most suitable reference gene for studies associated with the sheep mandibular condyle.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Military Stomatology, Department of Oral and Maxillofacial Surgery, School of Stomatology, The Fourth Military Medical University, Xi'an, Shaanxi 710032, P.R. China.

ABSTRACT
Reference genes are commonly used as a reliable approach to normalize the results of quantitative polymerase chain reaction (qPCR), and to reduce errors in the relative quantification of gene expression. Suitable reference genes belonging to numerous functional classes have been identified for various types of species and tissue. However, little is currently known regarding the most suitable reference genes for bone, specifically for the sheep mandibular condyle. Sheep are important for the study of human bone diseases, particularly for temporomandibular diseases. The present study aimed to identify a set of reference genes suitable for the normalization of qPCR data from the mandibular condyle of sheep. A total of 12 reference genes belonging to various functional classes were selected, and the expression stability of the reference genes was determined in both the normal and fractured area of the sheep mandibular condyle. RefFinder, which integrates the following currently available computational algorithms: geNorm, NormFinder, BestKeeper, and the comparative ΔCt method, was used to compare and rank the candidate reference genes. The results obtained from the four methods demonstrated a similar trend: RPL19, ACTB, and PGK1 were the most stably expressed reference genes in the sheep mandibular condyle. As determined by RefFinder comprehensive analysis, the results of the present study suggested that RPL19 is the most suitable reference gene for studies associated with the sheep mandibular condyle. In addition, ACTB and PGK1 may be considered suitable alternatives.

Show MeSH
Related in: MedlinePlus