Evaluation of Internal Reference Genes for Quantitative Expression Analysis by Real-Time PCR in Ovine Whole Blood
| Autor: | Maria Grazia Maniaci, Claudio Bellino, Chiara Campanella, Dario Ariello, Simone Peletto, Antonio Quasso, Paola Modesto, Pier Luigi Acutis, Simone Bertuzzi, Maria Caramelli |
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| Rok vydání: | 2011 |
| Předmět: |
Ovis aries
SDHA Gene Expression Sheep Diseases reference genes real-time qPCR Biology Real-Time Polymerase Chain Reaction Catalysis Article lcsh:Chemistry Inorganic Chemistry Reference genes Gene expression Animals Physical and Theoretical Chemistry Selection Genetic reference genes Ovis aries whole blood real-time qPCR normalization lcsh:QH301-705.5 Molecular Biology Gene Spectroscopy DNA Primers Genetics Sheep Gene Expression Profiling whole blood normalization Organic Chemistry General Medicine Sequence Analysis DNA Reference Standards Computer Science Applications Gene expression profiling Real-time polymerase chain reaction lcsh:Biology (General) lcsh:QD1-999 Expression quantitative trait loci YWHAZ |
| Zdroj: | International Journal of Molecular Sciences International Journal of Molecular Sciences; Volume 12; Issue 11; Pages: 7732-7747 International Journal of Molecular Sciences, Vol 12, Iss 11, Pp 7732-7747 (2011) |
| ISSN: | 1422-0067 |
| DOI: | 10.3390/ijms12117732 |
| Popis: | The use of reference genes is commonly accepted as the most reliable approach to normalize qRT-PCR and to reduce possible errors in the quantification of gene expression. The most suitable reference genes in sheep have been identified for a restricted range of tissues, but no specific data on whole blood are available. The aim of this study was to identify a set of reference genes for normalizing qRT-PCR from ovine whole blood. We designed 11 PCR assays for commonly employed reference genes belonging to various functional classes and then determined their expression stability in whole blood samples from control and disease-stressed sheep. SDHA and YWHAZ were considered the most suitable internal controls as they were stably expressed regardless of disease status according to both geNorm and NormFinder software; furthermore, geNorm indicated SDHA/HPRT, YWHAZ/GAPDH and SDHA/YWHAZ as the best reference gene combinations in control, disease-stressed and combined sheep groups, respectively. Our study provides a validated panel of optimal control genes which may be useful for the identification of genes differentially expressed by qRT-PCR in a readily accessible tissue, with potential for discovering new physiological and disease markers and as a tool to improve production traits (e.g., by identifying expression Quantitative Trait Loci). An additional outcome of the study is a set of intron-spanning primer sequences suitable for gene expression experiments employing SYBR Green chemistry on other ovine tissues and cells. |
| Databáze: | OpenAIRE |
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