Abstract
Real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) is a powerful method for measurement of gene expression for diagnostic and prognostic studies of non-Hodgkin's lymphomas (NHL). In order for this technique to gain wide applicability, it is critically important to establish a uniform method for normalization of RNA input. In this study, we have determined the best method to quantify the RNA/cDNA input per reaction and searched for the most useful endogenous control genes for normalization of the measurements, based on their abundance and lowest variability between different types of lymphoid cells. To accomplish these aims, we have analyzed the RNA expression of 11 potential endogenous control genes (glyceraldehyde-3-phosphate dehydrogenase, β-actin, peptidylprolyl isomerase A, β2 microglobulin, protein kinase cGMP-dependent, type I, hypoxanthine phosphoribosyltransferase 1, TATA box binding protein, transferrin receptor, large ribosomal protein, β-glucoronidase and 18S ribosomal RNA). In all, 12 different B- and T-cell lymphoma/leukemia cell lines, 80 B- and T-cell NHL specimens, and resting and activated normal B and T lymphocytes were screened. Normalization of the nucleic acid input by spectrophotometric OD260 measurement of RNA proved more reliable than spectrophotometric or fluorometric measurements of cDNA or than electrophoretic estimation of the ribosomal and mRNA fractions. The protein kinase cGMP-dependent, type I (PRKG1) and the TBP genes were expressed at common abundance and exhibited the lowest variability among the cell specimens. We suggest that for further lymphoma studies based on the real-time RT-PCR quantification of gene expression, that RNA input in each reaction be equalized between the specimens by spectrophotometric OD260 measurements. The expression of the gene of interest in different samples should be normalized by concomitant measurement of the PRKG1 and/or the TBP gene products.
References
- 1.
Heid CA, Stevens J, Livak KJ, Williams PM. Real time quantitative PCR. Genome Res 1996; 6: 986–994.
- 2.
Lossos IS, Jones KD, Warnke R, Natkunam Y, Kaizer H, Zehnder JL et al. The expression of a single gene, BCL-6, strongly predicts survival in patients with diffuse large B-cell lymphoma. Blood 2001; 98: 945–951.
- 3.
Bruggemann M, Droese J, Bolz I, Luth P, Pott C, von Neuhoff N et al. Improved assessment of minimal residual disease in B cell malignancies using fluorogenic consensus probes for real-time quantitative PCR. Leukemia 2000; 14: 1419–1425.
- 4.
Medeiros LJ, Hai S, Thomazy VA, Estalilla OC, Romaguera J, Luthra R. Real-time RT-PCR assay for quantifying cyclin D1 mRNA in B-cell NON-Hodgkin's lymphomas. Mod Pathol 2002; 15: 556–564.
- 5.
Maes B, Vanhentenrijk V, Wlodarska I, Cools J, Peeters B, Marynen P et al. The NPM–ALK and the ATIC–ALK fusion genes can be detected in non-neoplastic cells. Am J Pathol 2001; 158: 2185–2193.
- 6.
Snider JV, Wechser MA, Lossos IS. Human disease characterization: real-time quantitative PCR analysis of gene expression. Drug Discov Today 2001; 6: 1062–1067.
- 7.
Suzuki T, Higgins PJ, Crawford DR. Control selection for RNA quantitation. Biotechniques 2000; 29: 332–337.
- 8.
Bustin SA. Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol 2000; 25: 169–193.
- 9.
Baseggio L, Bienvenu J, Charlot C, Picollet J, Felman P, Coiffier B et al. Higher LPS-stimulated TNF-αmRNA levels in peripheral blood mononuclear cells from non-Hodgkin's lymphoma patients. Exp Hematol 2001; 29: 330–338.
- 10.
Bijwaard KE, Aguilera NS, Monczak Y, Trudel M, Taubenberger JK, Lichy JH. Quantitative real-time reverse transcription-PCR assay for cyclin D1 expression: utility in the diagnosis of mantle cell lymphoma. Clin Chem 2001; 47: 195–201.
- 11.
Husson H, Carideo EG, Neuberg D, Schultze J, Munoz O, Marks PW et al. Gene expression profiling of follicular lymphoma and normal germinal center B cells using cDNA arrays. Blood 2002; 99: 282–289.
- 12.
Harris NL, Jaffe ES, Stein H, Banks PM, Chan JK, Cleary ML et al. A revised European-American classification of lymphoid neoplasms: a proposal from the International Lymphoma Study Group [see comments]. Blood 1994; 84: 1361–1392.
- 13.
Alizadeh AA, Eisen MB, Davis RE, Ma C, Lossos IS, Rosenwald A et al. Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling. Nature 2000; 403: 503–511.
- 14.
Rosenwald A, Wright G, Chan WC, Connors JM, Campo E, Fisher RI et al. The use of molecular profiling to predict survival after chemotherapy for diffuse large-B-cell lymphoma. N Engl J Med 2002; 346: 1937–1947.
- 15.
Shipp MA, Ross KN, Tamayo P, Weng AP, Kutok JL, Aguiar RC et al. Diffuse large B-cell lymphoma outcome prediction by gene-expression profiling and supervised machine learning. Nat Med 2002; 8: 68–74.
- 16.
Feroze-Merzoug F, Berquin IM, Dey J, Chen YQ. Peptidylprolyl isomerase A (PPIA) as a preferred internal control over GAPDH and β-actin in quantitative RNA analyses. Biotechniques 2002; 32: 776–778,780,782.
- 17.
Finnegan MC, Goepel JR, Hancock BW, Goyns MH. Investigation of the expression of housekeeping genes in non-Hodgkin's lymphoma. Leukemia Lymphoma 1993; 10: 387–393.
- 18.
Lossos IS, Alizadeh AA, Diehn M, Warnke R, Thorstenson Y, Oefner PJ et al. Transformation of follicular lymphoma to diffuse large-cell lymphoma: alternative patterns with increased or decreased expression of c-myc and its regulated genes. Proc Natl Acad Sci USA 2002; 99: 8886–8891.
- 19.
Aviles A, Zepeda G, Diaz-Maqueo JC, Rodriguez L, Guzman R, Garcia EL et al. β2 microglobulin level as an indicator of prognosis in diffuse large cell lymphoma. Leukemia Lymphoma 1992; 7: 135–138.
- 20.
Ripple MO, Wilding G. Alteration of glyceraldehyde-3-phosphate dehydrogenase activity and messenger RNA content by androgen in human prostate carcinoma cells. Cancer Res 1995; 55: 4234–4236.
- 21.
Mansur NR, Meyer-Siegler K, Wurzer JC, Sirover MA. Cell cycle regulation of the glyceraldehyde-3-phosphate dehydrogenase/uracil DNA glycosylase gene in normal human cells. Nucleic Acids Res 1993; 21: 993–998.
- 22.
Noguchi I, Arai H, Iizuka R. A study on postmortem stability of vasopressin messenger RNA in rat brain compared with those in total RNA and ribosomal RNA. J Neural Transm Gen Sect 1991; 83: 171–178.
- 23.
Santiago TC, Purvis IJ, Bettany AJ, Brown AJ. The relationship between mRNA stability and length in Saccharomyces cerevisiae. Nucleic Acids Res 1986; 14: 8347–8360.
Acknowledgements
This work was supported by Grants CA33399 and CA34233 from the USPHS-NIH.
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Division of Oncology, Department of Medicine Stanford University Medical Center, Stanford, CA, USA
- I S Lossos
- , D K Czerwinski
- & R Levy
Applied Biosystems, Lincoln Center Drive, Foster City, CA, USA
- M A Wechser
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Correspondence to R Levy.
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