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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 geNorm. (Y-axis, stability values of the reference genes).
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f1-mmr-12-04-5633: Candidate reference genes ranked according to their expression stability, as determined by geNorm. (Y-axis, stability values of the reference genes).

Mentions: The stability values (M) of the reference genes were calculated using the geNorm algorithm. High M-values indicated high gene expression variability, whereas the most stable genes exhibited M-values <1.5. The M-values of the reference genes in the normal and fractured area of the sheep mandibular condyle as determined by geNorm, are shown in Table II and Fig. 1. In the control group, the most stable genes were RPL19 and ACTB, with an average M-value of 0.754. The second most stable gene was PGK1, with an average M-value of 0.948, whereas the least stable genes were SDHA and G6PD. However, in the test group, the most stable genes were S24 and B2M with an average M-value of 1.657, whereas the least stable genes were HPRT and YWHAZ. A comprehensive analysis of the combined group by geNorm identified the most stable genes to be RPL19 and ACTB, with an average M-value of 1.081. The second most stable gene in the combined group was PGK1, with an average M-value of 1.359. The remaining genes exhibited high M-values >1.5, with the least stable genes being SDHA and YWHAZ. Notably, SDHA and YWHAZ are considered conventional housekeeping genes in ovine blood (12).


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 geNorm. (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

f1-mmr-12-04-5633: Candidate reference genes ranked according to their expression stability, as determined by geNorm. (Y-axis, stability values of the reference genes).
Mentions: The stability values (M) of the reference genes were calculated using the geNorm algorithm. High M-values indicated high gene expression variability, whereas the most stable genes exhibited M-values <1.5. The M-values of the reference genes in the normal and fractured area of the sheep mandibular condyle as determined by geNorm, are shown in Table II and Fig. 1. In the control group, the most stable genes were RPL19 and ACTB, with an average M-value of 0.754. The second most stable gene was PGK1, with an average M-value of 0.948, whereas the least stable genes were SDHA and G6PD. However, in the test group, the most stable genes were S24 and B2M with an average M-value of 1.657, whereas the least stable genes were HPRT and YWHAZ. A comprehensive analysis of the combined group by geNorm identified the most stable genes to be RPL19 and ACTB, with an average M-value of 1.081. The second most stable gene in the combined group was PGK1, with an average M-value of 1.359. The remaining genes exhibited high M-values >1.5, with the least stable genes being SDHA and YWHAZ. Notably, SDHA and YWHAZ are considered conventional housekeeping genes in ovine blood (12).

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