Evaluation of real-time PCR endogenous control genes for analysis of gene expression in bovine endometrium

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Abstract

Background

Quantitative real-time PCR gene expression results are generally normalised using endogenous control genes. These reference genes should be expressed at a constant level across all sample groups in a study, and should not be influenced by study treatments or conditions. There has been no systematic investigation of endogenous control genes for bovine endometrium to date. The suitability of both commonly used and novel endogenous control genes was evaluated in this study, with the latter being selected from stably expressed transcripts identified through microarray analysis of bovine endometrium. Fifteen candidate endogenous control genes were assessed across different tissue subtypes in pregnant and cycling Holstein-Friesian dairy cows from two divergent genetic backgrounds.

Results

The expression profiles of five commonly used endogenous control genes (GAPDH, PPIA, RPS9, RPS15A, and UXT) and 10 experimentally derived candidate endogenous control genes (SUZ12, C2ORF29, ZNF131, ACTR1A, HDAC1, SLC30A6, CNOT7, DNAJC17, BBS2, and RANBP10) were analysed across 44 samples to determine the most stably expressed gene. Gene stability was assessed using the statistical algorithms GeNorm and Normfinder. All genes presented with low overall variability (0.87 to 1.48% CV of Cq). However, when used to normalise a differentially expressed gene (oxytocin receptor - OXTR) in the samples, the reported relative gene expression levels were significantly affected by the control gene chosen. Based on the results of this analysis, SUZ12 is proposed as the most appropriate control gene for use in bovine endometrium during early pregnancy or the oestrus cycle.

Conclusion

This study establishes the suitability of novel endogenous control genes for comparing expression levels in endometrial tissues of pregnant and cycling bovines, and demonstrates the utility of microarray analysis as a method for identifying endogenous control gene candidates.

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Most cited references 25

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Validation of housekeeping genes for normalizing RNA expression in real-time PCR.

Keertan Dheda, Jim Huggett, Stephen A. Bustin … (2004)

Analysis of RNA expression using techniques like real-time PCR has traditionally used reference or housekeeping genes to control for error between samples. This practice is being questioned as it becomes increasingly clear that some housekeeping genes may vary considerably in certain biological samples. We used real-time reverse transcription PCR (RT-PCR) to assess the levels of 13 housekeeping genes expressed in peripheral blood mononuclear cell culture and whole blood from healthy individuals and those with tuberculosis. Housekeeping genes were selected from conventionally used ones and from genes reported to be invariant in human T cell culture. None of the commonly used housekeeping genes [e.g., glyceraldehyde-phosphate-dehydrogenase (GAPDH)] were found to be suitable as internal references, as they were highly variable (>30-fold maximal variability). Furthermore, genes previously found to be invariant in human T cell culture also showed large variation in RNA expression (>34-fold maximal variability). Genes that were invariant in blood were highly variable in peripheral blood mononuclear cell culture. Our data show that RNA specifying human acidic ribosomal protein was the most suitable housekeeping gene for normalizing mRNA levels in human pulmonary tuberculosis. Validations of housekeeping genes are highly specific for a particular experimental model and are a crucial component in assessing any new model.

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Microarray validation: factors influencing correlation between oligonucleotide microarrays and real-time PCR

Jeanine S Morey, James C Ryan, Frances Van Dolah (2006)

Quantitative real-time PCR (qPCR) is a commonly used validation tool for confirming gene expression results obtained from microarray analysis; however, microarray and qPCR data often result in disagreement. The current study assesses factors contributing to the correlation between these methods in five separate experiments employing two-color 60-mer oligonucleotide microarrays and qPCR using SYBR green. Overall, significant correlation was observed between microarray and qPCR results (ρ=0.708, p 0.80 consistently observed when quality scores are applied.

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The power of real-time PCR.

Mark Valasek, J Repa (2005)

In recent years, real-time polymerase chain reaction (PCR) has emerged as a robust and widely used methodology for biological investigation because it can detect and quantify very small amounts of specific nucleic acid sequences. As a research tool, a major application of this technology is the rapid and accurate assessment of changes in gene expression as a result of physiology, pathophysiology, or development. This method can be applied to model systems to measure responses to experimental stimuli and to gain insight into potential changes in protein level and function. Thus physiology can be correlated with molecular events to gain a better understanding of biological processes. For clinical molecular diagnostics, real-time PCR can be used to measure viral or bacterial loads or evaluate cancer status. Here, we discuss the basic concepts, chemistries, and instrumentation of real-time PCR and include present applications and future perspectives for this technology in biomedical sciences and in life science education.

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Author and article information

Journal

Journal ID (nlm-ta): BMC Mol Biol

Title: BMC Molecular Biology

Publisher: BioMed Central

ISSN (Electronic): 1471-2199

Publication date Collection: 2009

Publication date (Electronic): 1 November 2009

Volume: 10

Page: 100

Affiliations

[1 ]DairyNZ Ltd., Hamilton, New Zealand

[2 ]Liggins Institute, The University of Auckland, Grafton, New Zealand

Article

Publisher ID: 1471-2199-10-100

DOI: 10.1186/1471-2199-10-100

PMC ID: 2774697

PubMed ID: 19878604

SO-VID: a26df5bf-ed6a-4392-ab32-96096f2ca101

License:

This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Evaluation of real-time PCR endogenous control genes for analysis of gene expression in bovine endometrium

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Abstract

Background

Results

Conclusion

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Genes & Diseases

Most cited references 25

Validation of housekeeping genes for normalizing RNA expression in real-time PCR.

Microarray validation: factors influencing correlation between oligonucleotide microarrays and real-time PCR

The power of real-time PCR.

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