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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 ranked according to their expression stability, as determined by BestKeeper. (Y-axis, stability values of the reference genes).
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f3-mmr-12-04-5633: Candidate reference genes ranked according to their expression stability, as determined by BestKeeper. (Y-axis, stability values of the reference genes).

Mentions: Analysis of the reference gene expression levels was carried out using the BestKeeper software (Table IV and Fig. 3). In the control group, the results showed that ACTB was the most stable gene, with a stability value of 0.78. In the test group, PGK1 was identified as the most stable reference gene, with a stability value of 0.93. In the combined group, PGK1 was determined to be the most stably expressed reference gene with a stability value of 0.96, whereas YWAZH was the least stable reference gene.


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 ranked according to their expression stability, as determined by BestKeeper. (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

f3-mmr-12-04-5633: Candidate reference genes ranked according to their expression stability, as determined by BestKeeper. (Y-axis, stability values of the reference genes).
Mentions: Analysis of the reference gene expression levels was carried out using the BestKeeper software (Table IV and Fig. 3). In the control group, the results showed that ACTB was the most stable gene, with a stability value of 0.78. In the test group, PGK1 was identified as the most stable reference gene, with a stability value of 0.93. In the combined group, PGK1 was determined to be the most stably expressed reference gene with a stability value of 0.96, whereas YWAZH was the least stable reference gene.

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