High-throughput identification of reference genes for research and clinical RT-qPCR analysis of breast cancer samples

Journal of Clinical Bioinformatics

Alexander G Tonevitsky²  Udo Schumacher¹  Vladimir V Galatenko⁴  Maxim U Shkurnikov²  Alexey E Lebedev⁴  Elona O Matveeva³  Nikita N Fedotov⁴  Nadezda A Khaustova³  Diana V Maltseva³ 

[1] Department of Anatomy II: Experimental Morphology, University Cancer Center Hamburg, University Medical Center Hamburg-Eppendorf, Hamburg, Germany;Institute of General Pathology and Pathophysiology, Moscow, Russia;SRC Bioclinicum, Moscow, Russia;Faculty of Mechanics and Mathematics of Lomonosov Moscow State University, Moscow, Russia

关键词: Breast cancer;    Gene expression;    Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR);    Microarrays;    Reference genes;   

Others  :  802834
DOI  :  10.1186/2043-9113-3-13

 received in 2013-03-08, accepted in 2013-07-12,  发布年份 2013

【 摘 要 】

Background

Quantification and normalization of RT-qPCR data critically depends on the expression of so called reference genes. Our goal was to develop a strategy for the selection of reference genes that utilizes microarray data analysis and combines known approaches for gene stability evaluation and to select a set of appropriate reference genes for research and clinical analysis of breast samples with different receptor and cancer status using this strategy.

Methods

A preliminary search of reference genes was based on high-throughput analysis of microarray datasets. The final selection and validation of the candidate genes were based on the RT-qPCR data analysis using several known methods for expression stability evaluation: comparative ∆Ct method, geNorm, NormFinder and Haller equivalence test.

Results

A set of five reference genes was identified: ACTB, RPS23, HUWE1, EEF1A1 and SF3A1. The initial selection was based on the analysis of publically available well-annotated microarray datasets containing different breast cancers and normal breast epithelium from breast cancer patients and epithelium from cancer-free patients. The final selection and validation were performed using RT-qPCR data from 39 breast cancer biopsy samples. Three genes from the final set were identified by the means of microarray analysis and were novel in the context of breast cancer assay. We showed that the selected set of reference genes is more stable in comparison not only with individual genes, but also with a system of reference genes used in commercial OncotypeDX test.

Conclusion

A selection of reference genes for RT-qPCR can be efficiently performed by combining a preliminary search based on the high-throughput analysis of microarray datasets and final selection and validation based on the analysis of RT-qPCR data with a simultaneous examination of different expression stability measures. The identified set of reference genes proved to be less variable and thus potentially more efficient for research and clinical analysis of breast samples comparing to individual genes and the set of reference genes used in OncotypeDX assay.

【 授权许可】

   
2013 Maltseva et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

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