Editorial Type:
Analytic Techniques

Array-based comparative genomic hybridization–guided identification of reference genes for normalization of real-time quantitative polymerase chain reaction assay data for lymphomas, histiocytic sarcomas, and osteosarcomas of dogs

Pei-Chien Tsai Department of Molecular Biomedical Science, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27606.

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 PhD
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Matthew Breen Department of Molecular Biomedical Science, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27606.
Center for Comparative Medicine and Translational Research, North Carolina State University, Raleigh, NC 27606.
Cancer Genetics Program, UNC Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599.

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DOI:
https://doi.org/10.2460/ajvr.73.9.1335
Volume/Issue:
Volume 73: Issue 9
Online Publication Date:
01 Sep 2012
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Abstract

Objective—To identify suitable reference genes for normalization of real-time quantitative PCR (RT-qPCR) assay data for common tumors of dogs.

Sample—Malignant lymph node (n = 8), appendicular osteosarcoma (9), and histiocytic sarcoma (12) samples and control samples of various nonneoplastic canine tissues.

Procedures—Array-based comparative genomic hybridization (aCGH) data were used to guide selection of 9 candidate reference genes. Expression stability of candidate reference genes and 4 commonly used reference genes was determined for tumor samples with RT-qPCR assays and 3 software programs.

Results—LOC611555 was the candidate reference gene with the highest expression stability among the 3 tumor types. Of the commonly used reference genes, expression stability of HPRT was high in histiocytic sarcoma samples, and expression stability of Ubi and RPL32 was high in osteosarcoma samples. Some of the candidate reference genes had higher expression stability than did the commonly used reference genes.

Conclusions and Clinical Relevance—Data for constitutively expressed genes with high expression stability are required for normalization of RT-qPCR assay results. Without such data, accurate quantification of gene expression in tumor tissue samples is difficult. Results of the present study indicated LOC611555 may be a useful RT-qPCR assay reference gene for multiple tissue types. Some commonly used reference genes may be suitable for normalization of gene expression data for tumors of dogs, such as lymphomas, osteosarcomas, or histiocytic sarcomas.

Abstract

Objective—To identify suitable reference genes for normalization of real-time quantitative PCR (RT-qPCR) assay data for common tumors of dogs.

Sample—Malignant lymph node (n = 8), appendicular osteosarcoma (9), and histiocytic sarcoma (12) samples and control samples of various nonneoplastic canine tissues.

Procedures—Array-based comparative genomic hybridization (aCGH) data were used to guide selection of 9 candidate reference genes. Expression stability of candidate reference genes and 4 commonly used reference genes was determined for tumor samples with RT-qPCR assays and 3 software programs.

Results—LOC611555 was the candidate reference gene with the highest expression stability among the 3 tumor types. Of the commonly used reference genes, expression stability of HPRT was high in histiocytic sarcoma samples, and expression stability of Ubi and RPL32 was high in osteosarcoma samples. Some of the candidate reference genes had higher expression stability than did the commonly used reference genes.

Conclusions and Clinical Relevance—Data for constitutively expressed genes with high expression stability are required for normalization of RT-qPCR assay results. Without such data, accurate quantification of gene expression in tumor tissue samples is difficult. Results of the present study indicated LOC611555 may be a useful RT-qPCR assay reference gene for multiple tissue types. Some commonly used reference genes may be suitable for normalization of gene expression data for tumors of dogs, such as lymphomas, osteosarcomas, or histiocytic sarcomas.

Contributor Notes

Supported in part by grants from the AKC Canine Health Foundation and the National Institutes of Health (awarded to Dr. Breen).

The authors thank Drs. Rachael Thomas, Andrea Angstadt, and Benoit Hedan for providing data for DNA copy number differences among canine lymphoma, osteosarcoma, and histiocytic sarcoma samples, respectively, and Drs. Steven Suter and Kristy Richards for providing some of the canine tissues and RNA samples.

Address correspondence to Dr. Breen (Matthew_Breen@ncsu.edu).
Cover American Journal of Veterinary Research
American Journal of Veterinary Research
Publication Date:
01 Sep 2012
  • 1.

    Vail DMMacEwen EG. Spontaneously occurring tumors of companion animals as models for human cancer. Cancer Invest 2000; 18:781–792.

  • 2.

    Vail DMYoung KM. Canine lymphoma and lymphoid leukemia. In: Withrow SJVail DM, eds. Small animal clinical oncology. 4th ed. Philadelphia: Elsevier Health Sciences, 2007:698–732.

    • Search Google Scholar
    • Export Citation
  • 3.

    Merlo DFRossi LPellegrino C, et al. Cancer incidence in pet dogs: findings of the Animal Tumor Registry of Genoa, Italy. J Vet Intern Med 2008; 22:976–984.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 4.

    Priester WAMcKay FW. The occurrence of tumors in domestic animals. Natl Cancer Inst Monogr 1980; 54:1–210.

  • 5.

    Bonnett BNEgenvall AOlson P, et al. Mortality in insured Swedish dogs: rates and causes of death in various breeds. Vet Rec 1997; 141:40–44.

  • 6.

    Lindblad-Toh KWade CMMikkelsen TS, et al. Genome sequence, comparative analysis and haplotype structure of the domestic dog. Nature 2005; 438:803–819.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 7.

    Heid CAStevens JLivak KJ, et al. Real time quantitative PCR. Genome Res 1996; 6:986–994.

  • 8.

    Higuchi RFockler CDollinger G, et al. Kinetic PCR analysis: real-time monitoring of DNA amplification reactions. Biotechnology (NY) 1993; 11:1026–1030.

    • Search Google Scholar
    • Export Citation
  • 9.

    Bustin SA. Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol 2000; 25:169–193.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 10.

    Thellin OZorzi WLakaye B, et al. Housekeeping genes as internal standards: use and limits. J Biotechnol 1999; 75:291–295.

  • 11.

    Bustin SA. Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. J Mol Endocrinol 2002; 29:23–39.

  • 12.

    Dheda KHuggett JFBustin SA, et al. Validation of housekeeping genes for normalizing RNA expression in real-time PCR. Biotechniques 2004; 37:112–119.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 13.

    Lockwood WWChari RChi B, et al. Recent advances in array comparative genomic hybridization technologies and their applications in human genetics. Eur J Hum Genet 2006; 14:139–148.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 14.

    Pinkel DAlbertson DG. Array comparative genomic hybridization and its applications in cancer. Nat Genet 2005; 37(suppl):S11–S17.

  • 15.

    Davies JJWilson IMLam WL. Array CGH technologies and their applications to cancer genomes. Chromosome Res 2005; 13:237–248.

  • 16.

    Fiegler HCarr PDouglas EJ, et al. DNA microarrays for comparative genomic hybridization based on DOP-PCR amplification of BAC and PAC clones. Genes Chromosomes Cancer 2003; 36:361–374.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 17.

    Thomas RScott ALangford CF, et al. Construction of a 2-Mb resolution BAC microarray for CGH analysis of canine tumors. Genome Res 2005; 15:1831–1837.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 18.

    Thomas RDuke SEKarlsson EK, et al. A genome assembly-integrated dog 1 Mb BAC microarray: a cytogenetic resource for canine cancer studies and comparative genomic analysis. Cytogenet Genome Res 2008; 122:110–121.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 19.

    Ishkanian ASMalloff CAWatson SK, et al. A tiling resolution DNA microarray with complete coverage of the human genome. Nat Genet 2004; 36:299–303.

  • 20.

    Greshock JNaylor TLMargolin A, et al. 1-Mb resolution array-based comparative genomic hybridization using a BAC clone set optimized for cancer gene analysis. Genome Res 2004; 14:179–187.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 21.

    Vissers LEde Vries BBOsoegawa K, et al. Array-based comparative genomic hybridization for the genomewide detection of submicroscopic chromosomal abnormalities. Am J Hum Genet 2003; 73:1261–1270.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 22.

    Snijders AMNowak NSegraves R, et al. Assembly of microarrays for genome-wide measurement of DNA copy number. Nat Genet 2001; 29:263–264.

  • 23.

    Etschmann BWilcken BStoevesand K, et al. Selection of reference genes for quantitative real-time PCR analysis in canine mammary tumors using the geNorm algorithm. Vet Pathol 2006; 43:934–942.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 24.

    Peters IRPeeters DHelps CR, et al. Development and application of multiple internal reference (housekeeper) gene assays for accurate normalisation of canine gene expression studies. Vet Immunol Immunopathol 2007; 117:55–66.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 25.

    Brinkhof BSpee BRothuizen J, et al. Development and evaluation of canine reference genes for accurate quantification of gene expression. Anal Biochem 2006; 356:36–43.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 26.

    Vandesompele JDe Preter KPattyn F, et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 2002; 3: RESEARCH0034.

    • Search Google Scholar
    • Export Citation
  • 27.

    Andersen CLJensen JLOrntoft TF. Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res 2004; 64:5245–5250.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 28.

    Pfaffl MWTichopad APrgomet C, et al. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper—Excel-based tool using pairwise correlations. Biotechnol Lett 2004; 26:509–515.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 29.

    Thomas RSeiser ELMotsinger-Reif AA, et al. Refining tumor-associated aneuploidy through ‘genomic recoding’ of recurrent DNA copy number aberrations in 150 canine non-Hodgkin's lymphomas. Leuk Lymphoma 2011; 52:1321–1335.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 30.

    Angstadt AYMotsinger-Reif AAThomas R, et al. Characterization of canine osteosarcoma by array comparative genomic hybridization and RT-qPCR: signatures of genomic imbalance in canine osteosarcoma parallel the human counterpart. Genes Chromosomes Cancer 2011; 50:859–874.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 31.

    Hedan BThomas RMotsinger-Reif AA, et al. Molecular cytogenetic characterization of canine histiocytic sarcoma: a spontaneous model for human histiocytic cancer identifies deletion of tumor suppressor genes and highlights influence of genetic background on tumor behavior. BMC Cancer 2011; 11:201.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 32.

    Rozen SSkaletsky HJ. Primer3 on the WWW for general users and for biologist programmers. In: Krawetz SMisener S, eds. Bioinformatics methods and protocols: methods in molecular biology. Totowa, NJ: Humana Press, 2000:365–386.

    • Search Google Scholar
    • Export Citation
  • 33.

    Maccoux LJClements DNSalway F, et al. Identification of new reference genes for the normalisation of canine osteoarthritic joint tissue transcripts from microarray data. BMC Mol Biol 2007; 8:62.

    • Crossref
    • Search Google Scholar
    • Export Citation

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