Molecular Biology

, 45:211 | Cite as

RPN1, a new reference gene for quantitative data normalization in lung and kidney cancer

  • G. S. Krasnov
  • N. Yu. Oparina
  • A. A. Dmitriev
  • A. V. Kudryavtseva
  • E. A. Anedchenko
  • T. T. Kondrat’eva
  • E. R. Zabarovsky
  • V. N. Senchenko
Genomics. Transcriptomics

Abstract

Quantitative methods of gene expression analysis in tumors require accurate data normalization, which allows comparison of different specimens with unknown mRNA/cDNA concentrations. For this purpose, reference genes with stable expression are used (e.g., GAPDH, ACTB, HPRT1, or TBP). The problem of choosing proper reference genes is still a topical issue, because well-known reference genes can be unsuitable for certain cancer types and their inappropriate use without additional testing can lead to wrong conclusions. A recently developed bioinformatical approach was employed to identify a new potential reference gene for lung and kidney tumors, RPN1, located on the long arm of chromosome 3. The method employed the mining of the dbEST and Oncomine databases and functional analysis of genes. RPN1 was selected from approximately 1500 candidate housekeeping genes. Using comparative genomic hybridization with NotI microarrays, we found no methylation, deletions, and/or amplifications in the RPN1-containing locus in 56 nonsmall cell lung and 42 clear cell renal cell cancer specimens. Real-time PCR showed that variation of RPN1 mRNA levels in nonsmall cell lung cancer and clear-cell renal cancer was low and comparable to that of the known reference genes GAPDH and GUSB, respectively. Expression levels of two hyalouronidase genes, HYAL1 and HYAL2, were assessed using the suggested references gene pairs (RPN1-GAPDH for lung cancer and RPN1-GUSB for kidney cancer), and these combinations were shown to produce accurate and reproducible data. These results suggest that RPN1 is a new, promising reference gene for quantitative data normalization in gene expression studies for lung and kidney cancers.

Keywords

reference genes normalization ribophorin nonsmall cell lung cancer clear cell renal cancer 

References

  1. 1.
    Lonneborg A., Aaroe J., Dumeaux V., Borresen-Dale A.L. 2009. Found in transcription: Gene expression and other novel blood biomarkers for the early detection of breast cancer. Expert. Rev. Anticancer Ther. 9, 1115–1123.PubMedCrossRefGoogle Scholar
  2. 2.
    Delgado M.D., Leon J. 2006. Gene expression regulation and cancer. Clin. Transl. Oncol. 8, 780–787.PubMedCrossRefGoogle Scholar
  3. 3.
    Livak K.J., Schmittgen T.D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the ΔΔCt method. Methods. 25, 402–408.PubMedCrossRefGoogle Scholar
  4. 4.
    Pfaffl M.W. 2001. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 29, e45.PubMedCrossRefGoogle Scholar
  5. 5.
    Vandesompele J., de Preter K., Pattyn F., Poppe B., van Roy N., de Paepe A., Speleman F. 2002. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 3, RESEARCH0034.PubMedCrossRefGoogle Scholar
  6. 6.
    Huggett J., Dheda K., Bustin S., Zumla A. 2005. Real-time RT-PCR normalisation: Strategies and considerations. Genes Immun. 6, 279–284.PubMedCrossRefGoogle Scholar
  7. 7.
    Skrzypski M. 2008. Quantitative reverse transcriptase real-time polymerase chain reaction (qRT-PCR) in translational oncology: Lung cancer perspective. Lung Cancer. 59, 147–154.PubMedCrossRefGoogle Scholar
  8. 8.
    Tricarico C., Pinzani P., Bianchi S., Paglierani M., Distante V., Pazzagli M., Bustin S.A., Orlando C. 2002. Quantitative real-time reverse transcription polymerase chain reaction: normalization to rRNA or single housekeeping genes is inappropriate for human tissue biopsies. Anal. Biochem. 309, 293–300.PubMedCrossRefGoogle Scholar
  9. 9.
    Lallemant B., Evrard A., Combescure C., Chapuis H., Chambon G., Raynal C., Reynaud C., Sabra O., Joubert D., Hollande F., Lallemant J.G., Lumbroso S., Brouillet J.P. 2009. Reference gene selection for head and neck squamous cell carcinoma gene expression studies. BMC Mol. Biol. 10, 78.PubMedCrossRefGoogle Scholar
  10. 10.
    Toegel S., Huang W., Piana C., Unger F.M., Wirth M., Goldring M.B., Gabor F., Viernstein H. 2007. Selection of reliable reference genes for qPCR studies on chondroprotective action. BMC Mol. Biol. 8, 13.PubMedCrossRefGoogle Scholar
  11. 11.
    Kidd M., Nadler B., Mane S., Eick G., Malfertheiner M., Champaneria M., Pfragner R., Modlin I. 2007. GeneChip, geNorm, and gastrointestinal tumors: Novel reference genes for real-time PCR. Physiol. Genomics. 30, 363–370.PubMedCrossRefGoogle Scholar
  12. 12.
    Hellemans J., Mortier G., de Paepe A., Speleman F., Vandesompele J. 2007. qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data. Genome Biol. 8, R19PubMedCrossRefGoogle Scholar
  13. 13.
    Pfaffl M.W., Tichopad A., Prgomet C., Neuvians T.P. 2004. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper-Excel-based tool using pair-wise correlations. Biotechnol. Lett. 26, 509–515.PubMedCrossRefGoogle Scholar
  14. 14.
    Andersen C.L., Jensen J.L., Orntoft T.F. 2004. 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. 64, 5245–5250.PubMedCrossRefGoogle Scholar
  15. 15.
    Liu D.W., Chen S.T., Liu H.P. 2005. Choice of endogenous control for gene expression in nonsmall cell lung cancer. Eur. Respir. J. 26, 1002–1008.PubMedCrossRefGoogle Scholar
  16. 16.
    Gresner P., Gromadzinska J., Wasowicz W. 2009. Reference genes for gene expression studies on non-small cell lung cancer. Acta Biochim. Pol. 56, 307–316.PubMedGoogle Scholar
  17. 17.
    Saviozzi S., Cordero F., Lo Iacono M., Novello S., Scagliotti G.V., Calogero R.A. 2006. Selection of suitable reference genes for accurate normalization of gene expression profile studies in non-small cell lung cancer. BMC Cancer. 6, 200.PubMedCrossRefGoogle Scholar
  18. 18.
    Glenn S.T., Jones C.A., Liang P., Kaushik D., Gross K.W., Kim H.L. 2007. Expression profiling of archival renal tumors by quantitative PCR to validate prognostic markers. Biotechniques. 43, 639–640, 642–643, 647.PubMedCrossRefGoogle Scholar
  19. 19.
    Jung M., Ramankulov A., Roigas J., Johannsen M., Ringsdorf M., Kristiansen G., Jung K. 2007. In search of suitable reference genes for gene expression studies of human renal cell carcinoma by real-time PCR. BMC Mol. Biol. 8, 47.PubMedCrossRefGoogle Scholar
  20. 20.
    Kudriavtseva A.V., Anedchenko E.A., Oparina N.Yu., Krasnov G.S., Kashkin K.N., Dmitriev A.A., Zborovskaya I.B., Kondratjeva T.T., Vinogradova E.V., Zinovyeva M.V., Kopantsev E.P., Senchenko V.N. 2009. Expression of FTL and FTH genes encoding ferritin subunits in lung and renal carcinomas. Mol. Biol. (Moscow). 43, 972–981.CrossRefGoogle Scholar
  21. 21.
    Anedchenko E.A., Dmitriev A.A., Krasnov G.S., Kondratieva T.T., Kopantsev E.P., Vinogradova T.V., Zinovyeva M.V., Zborovskaya I.B., Polotsky B.E., Sakharova O.V., Kashuba V.I., Zabarovsky E.R., Senchenko V.N. 2008. Downregulation of RBSR3/CTDSRL, NRRL2/G21, RASSF1A, ITGA9, HYAL1, and HYAL2 in non-small cell lung cancer. Mol. Biol. (Moscow). 42, 859–869.CrossRefGoogle Scholar
  22. 22.
    Senchenko V.N., Anedchenko E.A., Kondratieva T.T., Krasnov G.S., Dmitriev A.A., Zabarovska V.I., Pavlova T.V., Kashuba V.I., Lerman M.I., Zabarovsky E.R. 2010. Simultaneous down-regulation of tumor suppressor genes RBSP3/CTDSPL, NPRL2/G21, and RASSF1A in primary non-small cell lung cancer. BMC Cancer. 10, 75.PubMedCrossRefGoogle Scholar
  23. 23.
  24. 24.
    University of California Santa Cruz; http://www.genome.ucsc.edu/
  25. 25.
    She X., Rohl C.A., Castle J.C., Kulkarni A.V., Johnson J.M., Chen R. 2009. Definition, conservation and epigenetics of housekeeping and tissue-enriched genes. BMC Genomics. 10, 269.PubMedCrossRefGoogle Scholar
  26. 26.
    Rhodes D.R., Yu J., Shanker K., Deshpande N., Varambally R., Ghosh D., Barrette T., Pandey A., Chinnaiyan A.M. 2004. ONCOMINE: A cancer microarray database and integrated data-mining platform. Neoplasia. 6, 1–6.PubMedGoogle Scholar
  27. 27.
  28. 28.
    Mitelman Database of Chromosome Aberrations and Gene Fusions in Cancer; http://cgap.nci.nih.gov/Chromosomes/Mitelman
  29. 29.
    Al-Shahrour F., Carbonell J., Minguez P., Goetz S., Conesa A., Tarraga J., Medina I., Alloza E., Montaner D., Dopazo J. 2008. Babelomics: advanced functional profiling of transcriptomics, proteomics and genomics experiments. Nucleic Acids Res. 36, W341–W346.PubMedCrossRefGoogle Scholar
  30. 30.
    Pavlova T.V., Kashuba V.I., Muravenko O.V., Yenamandra P.S., Ivanova T.A., Zabarovska V.I., Pakhmanaliev E.R., Petrenko L.A., Pronina I.V., Loginova V.I., Yurkevich O.Yu., Kisselev L.L., Zelenin A.V., Zabarovsky E.R. 2009. Use of NotI microarrays in analysis of epigenetic and structural changes in epithelial tumor genomes by the example of human chromosome 3. Mol. Biol. (Moscow). 43, 313–320.CrossRefGoogle Scholar
  31. 31.
    Anedchenko E.A., Kisseljova N.P., Dmitriev A.A., Kisseljov F.L., Zabarovsky E.R., Senchenko V.N. 2007. Tumor suppressor gene RBSR3 in cervical carcinoma: Copy number and transcription level. Mol. Biol. (Moscow). 41, 77–85.CrossRefGoogle Scholar
  32. 32.
    Wang F., Grigorieva E.V., Li J., Senchenko V.N., Pavlova T.V., Anedchenko E.A., Kudryavtseva A.V., Tsimanis A., Angeloni D., Lerman M.I., Kashuba V.I., Klein G., Zabarovsky E.R. 2008. HYAL1 and HYAL2 inhibit tumour growth in vivo but not in vitro. PLoS One. 3, e3031.PubMedCrossRefGoogle Scholar
  33. 33.
    Wilson C.M., Kraft C., Duggan C., Ismail N., Crawshaw S.G., High S. 2005. Ribophorin I associates with a subset of membrane proteins after their integration at the sec61 translocon. J. Biol. Chem. 280, 4195–4206.PubMedCrossRefGoogle Scholar
  34. 34.
    Wilson C.M., High S. 2007. Ribophorin I acts as a substrate-specific facilitator of N-glycosylation. J. Cell. Sci. 120, 648–657.PubMedCrossRefGoogle Scholar
  35. 35.
    Wilson C.M., Roebuck Q., High S. 2008. Ribophorin I regulates substrate delivery to the oligosaccharyltransferase core. Proc. Natl. Acad. Sci. U.S.A. 105, 9534–9539.PubMedCrossRefGoogle Scholar
  36. 36.
    Sanchez-Carbayo M., Socci N.D., Lozano J., Saint F., Cordon-Cardo C. 2006. Defining molecular profiles of poor outcome in patients with invasive bladder cancer using oligonucleotide microarrays. J. Clin. Oncol. 24, 778–789.PubMedCrossRefGoogle Scholar
  37. 37.
    Welsh J.B., Zarrinkar P.P., Sapinoso L.M., Kern S.G., Behling C.A., Monk B.J., Lockhart D.J., Burger R.A., Hampton G.M. 2001. Analysis of gene expression profiles in normal and neoplastic ovarian tissue samples identifies candidate molecular markers of epithelial ovarian cancer. Proc. Natl. Acad. Sci. U.S.A. 98, 1176–1181.PubMedCrossRefGoogle Scholar
  38. 38.
    Welsh J.B., Sapinoso L.M., Su A.I., Kern S.G., Wang-Rodriguez J., Moskaluk C.A., Frierson H.F. Jr., Hampton G.M. 2001. Analysis of gene expression identifies candidate markers and pharmacological targets in prostate cancer. Cancer Res. 61, 5974–5978.PubMedGoogle Scholar
  39. 39.
    Valk P.J., Verhaak R.G., Beijen M.A., Erpelinck C.A., Barjesteh van Waalwijk van Doorn-Khosrovani S., Boer J.M., Beverloo H.B., Moorhouse M.J., van der Spek P.J., Lowenberg B., Delwel R. 2004. Prognostically useful gene-expression profiles in acute myeloid leukemia. N. Engl. J. Med. 350, 1617–1628.PubMedCrossRefGoogle Scholar
  40. 40.
    Demirhan O., Tastemir D., Hasturk S., Kuleci S., Hanta I. 2010. Alterations in p16 and p53 genes and chromosomal findings in patients with lung cancer: Fluorescence in situ hybridization and cytogenetic studies. Cancer Epidemiol. 34, 472–477.PubMedCrossRefGoogle Scholar
  41. 41.
    Gronbaek K., Hother C., Jones P.A. 2007. Epigenetic changes in cancer. Acta Pathol. Microbiol. Immuniol. Scand. 115, 1039–1059.Google Scholar

Copyright information

© Pleiades Publishing, Ltd. 2011

Authors and Affiliations

  • G. S. Krasnov
    • 1
  • N. Yu. Oparina
    • 1
  • A. A. Dmitriev
    • 1
  • A. V. Kudryavtseva
    • 1
  • E. A. Anedchenko
    • 1
  • T. T. Kondrat’eva
    • 2
  • E. R. Zabarovsky
    • 3
  • V. N. Senchenko
    • 1
  1. 1.Engelhardt Institute of Molecular BiologyRussian Academy of SciencesMoscowRussia
  2. 2.Blokhin Cancer Research CenterRussian Academy of Medical SciencesMoscowRussia
  3. 3.MTCKarolinska InstituteStockholmSweden

Personalised recommendations