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Evidence based selection of commonly used RT-qPCR reference genes for the analysis of mouse skeletal muscle.

Thomas KC, Zheng XF, Garces Suarez F, Raftery JM, Quinlan KG, Yang N, North KN, Houweling PJ - PLoS ONE (2014)

Bottom Line: However, commonly used reference genes are often poorly validated and may change as a result of genetic background, environment and experimental intervention.Analysis of our experimental variant (Actn3 KO) also resulted in an altered ranking of reference gene suitability.Furthermore we demonstrate that a poor reference gene results in increased variability in the normalized expression of a gene of interest, and can result in loss of significance.

View Article: PubMed Central - PubMed

Affiliation: Institute for Neuroscience and Muscle Research, Children's Hospital at Westmead, Sydney, NSW, Australia.

ABSTRACT
The ability to obtain accurate and reproducible data using quantitative real-time Polymerase Chain Reaction (RT-qPCR) is limited by the process of data normalization. The use of 'housekeeping' or 'reference' genes is the most common technique used to normalize RT-qPCR data. However, commonly used reference genes are often poorly validated and may change as a result of genetic background, environment and experimental intervention. Here we present an analysis of 10 reference genes in mouse skeletal muscle (Actb, Aldoa, Gapdh, Hprt1, Ppia, Rer1, Rn18s, Rpl27, Rpl41 and Rpl7L1), which were identified as stable either by microarray or in the literature. Using the MIQE guidelines we compared wild-type (WT) mice across three genetic backgrounds (R129, C57BL/6j and C57BL/10) as well as analyzing the α-actinin-3 knockout (Actn3 KO) mouse, which is a model of the common polymorphism (R577X) in human ACTN3. Comparing WT mice across three genetic backgrounds, we found that different genes were more tightly regulated in each strain. We have developed a ranked profile of the top performing reference genes in skeletal muscle across these common mouse strains. Interestingly the commonly used reference genes; Gapdh, Rn18s, Hprt1 and Actb were not the most stable. Analysis of our experimental variant (Actn3 KO) also resulted in an altered ranking of reference gene suitability. Furthermore we demonstrate that a poor reference gene results in increased variability in the normalized expression of a gene of interest, and can result in loss of significance. Our data demonstrate that reference genes need to be validated prior to use. For the most accurate normalization, it is important to test several genes and use the geometric mean of at least three of the most stably expressed genes. In the analysis of mouse skeletal muscle, strain and intervention played an important role in selecting the most stable reference genes.

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Related in: MedlinePlus

RefFinder analysis comparing the selected reference genes in WT and Actn3 KO mice.A) R129 WT and Actn3 KO mice, B) C57BL/6j WT and Actn3 KO mice, C) C57BL/10 WT and Actn3 KO mice. Columns are shaded light to dark based on R129 gene stability order to represent a shift in position between the C57BL/6j and C57BL/10 strains. 1 – 3 represent the top three genes in each analysis.
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pone-0088653-g003: RefFinder analysis comparing the selected reference genes in WT and Actn3 KO mice.A) R129 WT and Actn3 KO mice, B) C57BL/6j WT and Actn3 KO mice, C) C57BL/10 WT and Actn3 KO mice. Columns are shaded light to dark based on R129 gene stability order to represent a shift in position between the C57BL/6j and C57BL/10 strains. 1 – 3 represent the top three genes in each analysis.

Mentions: To examine how experimental intervention affects the appropriateness of a reference gene, we analysed a common skeletal muscle variant (Actn3 KO) and looked at reference gene stability between the Actn3 WT and KO mice across three different mouse strains. The stability of selected genes for each mouse strain (R129, C57BL/6j and C57BL/10) is shown in Figure 3. In this case the stability value is a measure of the effect of α-actinin-3 deficiency on the expression of the reference gene, with lower numbers reflecting a minimal effect highlighting the most stable genes for use with this particular genetic modification.


Evidence based selection of commonly used RT-qPCR reference genes for the analysis of mouse skeletal muscle.

Thomas KC, Zheng XF, Garces Suarez F, Raftery JM, Quinlan KG, Yang N, North KN, Houweling PJ - PLoS ONE (2014)

RefFinder analysis comparing the selected reference genes in WT and Actn3 KO mice.A) R129 WT and Actn3 KO mice, B) C57BL/6j WT and Actn3 KO mice, C) C57BL/10 WT and Actn3 KO mice. Columns are shaded light to dark based on R129 gene stability order to represent a shift in position between the C57BL/6j and C57BL/10 strains. 1 – 3 represent the top three genes in each analysis.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3921188&req=5

pone-0088653-g003: RefFinder analysis comparing the selected reference genes in WT and Actn3 KO mice.A) R129 WT and Actn3 KO mice, B) C57BL/6j WT and Actn3 KO mice, C) C57BL/10 WT and Actn3 KO mice. Columns are shaded light to dark based on R129 gene stability order to represent a shift in position between the C57BL/6j and C57BL/10 strains. 1 – 3 represent the top three genes in each analysis.
Mentions: To examine how experimental intervention affects the appropriateness of a reference gene, we analysed a common skeletal muscle variant (Actn3 KO) and looked at reference gene stability between the Actn3 WT and KO mice across three different mouse strains. The stability of selected genes for each mouse strain (R129, C57BL/6j and C57BL/10) is shown in Figure 3. In this case the stability value is a measure of the effect of α-actinin-3 deficiency on the expression of the reference gene, with lower numbers reflecting a minimal effect highlighting the most stable genes for use with this particular genetic modification.

Bottom Line: However, commonly used reference genes are often poorly validated and may change as a result of genetic background, environment and experimental intervention.Analysis of our experimental variant (Actn3 KO) also resulted in an altered ranking of reference gene suitability.Furthermore we demonstrate that a poor reference gene results in increased variability in the normalized expression of a gene of interest, and can result in loss of significance.

View Article: PubMed Central - PubMed

Affiliation: Institute for Neuroscience and Muscle Research, Children's Hospital at Westmead, Sydney, NSW, Australia.

ABSTRACT
The ability to obtain accurate and reproducible data using quantitative real-time Polymerase Chain Reaction (RT-qPCR) is limited by the process of data normalization. The use of 'housekeeping' or 'reference' genes is the most common technique used to normalize RT-qPCR data. However, commonly used reference genes are often poorly validated and may change as a result of genetic background, environment and experimental intervention. Here we present an analysis of 10 reference genes in mouse skeletal muscle (Actb, Aldoa, Gapdh, Hprt1, Ppia, Rer1, Rn18s, Rpl27, Rpl41 and Rpl7L1), which were identified as stable either by microarray or in the literature. Using the MIQE guidelines we compared wild-type (WT) mice across three genetic backgrounds (R129, C57BL/6j and C57BL/10) as well as analyzing the α-actinin-3 knockout (Actn3 KO) mouse, which is a model of the common polymorphism (R577X) in human ACTN3. Comparing WT mice across three genetic backgrounds, we found that different genes were more tightly regulated in each strain. We have developed a ranked profile of the top performing reference genes in skeletal muscle across these common mouse strains. Interestingly the commonly used reference genes; Gapdh, Rn18s, Hprt1 and Actb were not the most stable. Analysis of our experimental variant (Actn3 KO) also resulted in an altered ranking of reference gene suitability. Furthermore we demonstrate that a poor reference gene results in increased variability in the normalized expression of a gene of interest, and can result in loss of significance. Our data demonstrate that reference genes need to be validated prior to use. For the most accurate normalization, it is important to test several genes and use the geometric mean of at least three of the most stably expressed genes. In the analysis of mouse skeletal muscle, strain and intervention played an important role in selecting the most stable reference genes.

Show MeSH
Related in: MedlinePlus