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Validation of internal reference genes for quantitative real-time PCR in a non-model organism, the yellow-necked mouse, Apodemus flavicollis.

ABSTRACT: BACKGROUND: Reference genes are used as internal standards to normalize mRNA abundance in quantitative real-time PCR and thereby allow a direct comparison between samples. So far most of these expression studies used human or classical laboratory model species whereas studies on non-model organism under in-situ conditions are quite rare. However, only studies in free-ranging populations can reveal the effects of natural selection on the expression levels of functional important genes. In order to test the feasibility of gene expression studies in wildlife samples we transferred and validated potential reference genes that were developed for lab mice (Mus musculus) to samples of wild yellow-necked mice, Apodemus flavicollis. The stability and suitability of eight potential reference genes was accessed by the programs BestKeeper, NormFinder and geNorm. FINDINGS: Although the three programs used different algorithms the ranking order of reference genes was significantly concordant and geNorm differed in only one, NormFinder in two positions compared to BestKeeper. The genes ordered by their mean rank from the most to the least stable gene were: Rps18, Sdha, Canx, Actg1, Pgk1, Ubc, Rpl13a and Actb. Analyses of the normalization factor revealed best results when the five most stable genes were included for normalization. DISCUSSION: We established a SYBR green qPCR assay for liver samples of wild A. flavicollis and conclude that five genes should be used for appropriate normalization. Our study provides the basis to investigate differential expression of genes under selection under natural selection conditions in liver samples of A. flavicollis. This approach might also be applicable to other non-model organisms.

SUBMITTER: Axtner J

PROVIDER: S-EPMC2804578 | biostudies-literature | 2009

REPOSITORIES: biostudies-literature

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Validation of internal reference genes for quantitative real-time PCR in a non-model organism, the yellow-necked mouse, Apodemus flavicollis.

Axtner Jan J Sommer Simone S

BMC research notes 20091223

<h4>Background</h4>Reference genes are used as internal standards to normalize mRNA abundance in quantitative real-time PCR and thereby allow a direct comparison between samples. So far most of these expression studies used human or classical laboratory model species whereas studies on non-model organism under in-situ conditions are quite rare. However, only studies in free-ranging populations can reveal the effects of natural selection on the expression levels of functional important genes. In ...[more]

PMID: 20030847

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Project description:BackgroundMammalian mandible and cranium are well-established model systems for studying canalization and developmental stability (DS) as two elements of developmental homeostasis. Nematode infections are usually acquired in early life and increase in intensity with age, while canalization and DS of rodent skulls could vary through late postnatal ontogeny. We aimed to estimate magnitudes and describe patterns of mandibular and cranial canalization and DS related to age and parasite intensity (diversity) in adult yellow-necked mice (Apodemus flavicollis).ResultsWe found the absence of age-related changes in the levels of canalization for mandibular and cranial size and DS for mandibular size. However, individual measures of mandibular and cranial shape variance increased, while individual measures of mandibular shape fluctuating asymmetry (FA) decreased with age. We detected mandibular and cranial shape changes during postnatal ontogeny, but revealed no age-related dynamics of their covariance structure among and within individuals. Categories regarding parasitism differed in the level of canalization for cranial size and the level of DS for cranial shape. We observed differences in age-related dynamics of the level of canalization between non-parasitized and parasitized animals, as well as between yellow-necked mice parasitized by different number of nematode species. Likewise, individual measures of mandibular and cranial shape FA decreased with age for the mandible in the less parasitized category and increased for the cranium in the most parasitized category.ConclusionsOur age-related results partly agree with previous findings. However, no rodent study so far has explored age-related changes in the magnitude of FA for mandibular size or mandibular and cranial FA covariance structure. This is the first study dealing with the nematode parasitism-related canalization and DS in rodents. We showed that nematode parasitism does not affect mandibular and cranial shape variation and covariance structure among and within individuals. However, parasite intensity (diversity) is related to ontogenetic dynamics of the levels of canalization and DS. Overall, additional studies on animals from natural populations are required before drawing some general conclusions.

Dataset Information

Validation of internal reference genes for quantitative real-time PCR in a non-model organism, the yellow-necked mouse, Apodemus flavicollis.

Publications

Validation of internal reference genes for quantitative real-time PCR in a non-model organism, the yellow-necked mouse, Apodemus flavicollis.

Similar Datasets

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