BACKGROUND: For accuracy of quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), normalisation with suitable reference genes is required. To date, no reference genes have been validated for expression studies of bronchoalveolar (BAL) cells. The aims of this study were to identify gene(s) with stable mRNA expression in BAL cells irrespective of gender, smoking, BAL cellular composition, lung pathology, treatment; and to assess the influence of reference genes on target gene expression data. RESULTS: The mRNA expression of ten housekeeping genes (ACTB, ARF1, CANX, G6PD, GAPDH, GPS1, GNB2L1, PSMB2, PSMD2, RPL32) was investigated by qRT-PCR in BAL cells from 71 subjects across a spectrum of lung diseases. The analyses were validated in an independent BAL cohort from 63 sarcoidosis patients and 17 control subjects. A second derivative method was used to calculate expression values (CTt); an equivalence test, applets BestKeeper, geNorm and NormFinder were applied to investigate gene expression stability. Of the investigated genes, PSMB2 (CTt +/- SD, 23.66 +/- 0.86) and RPL32 (18.65 +/- 0.92) were the most stable; both were constantly expressed in BAL samples from parallel investigated cohorts irrespective of evaluated variables. Finally, to demonstrate effect of traditional (ACTB/GAPDH) and novel (PSMB2/RPL32) reference genes as denominators, expression of two cytokines known associated with sarcoidosis was investigated in sarcoid BAL cells. While normalization with PSMB2/RPL32 resulted in elevated IFNG mRNA expression (p = 0.004); no change was observed using GAPDH/ACTB (p > 0.05). CCL2 mRNA up-regulation was observed only when PSMB2/RPL32 were used as denominators (p < 0.03). CONCLUSION: PSMB2 and RPL32 are, therefore, suitable reference genes to normalize qRT-PCR in BAL cells in sarcoidosis, and other interstitial lung disease.

Department of Immunology Palacky University The Czech Republic

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Dheda K, Huggett JF, Bustin SA, Johnson MA, Rook G, Zumla A. Validation of housekeeping genes for normalizing RNA expression in real-time PCR. Biotechniques. 2004;37:112–4. PubMed

Bustin SA, Benes V, Nolan T, Pfaffl MW. Quantitative real-time RT-PCR-a perspective. J Mol Endocrinol. 2005;34:597–601. doi: 10.1677/jme.1.01755. PubMed DOI

Huggett J, Dheda K, Bustin S, Zumla A. Real-time RT-PCR normalization; strategies and considerations. Genes Immun. 2005;6:279–84. doi: 10.1038/sj.gene.6364190. PubMed DOI

Studer SM, Kaminski N. Towards systems biology of human pulmonary fibrosis. Proc Am Thorac Soc. 2007;4:85–91. doi: 10.1513/pats.200607-139JG. PubMed DOI PMC

Bustin SA, Mueller R. Real-time reverse transcription PCR (qRT-PCR) and its potential use in clinical diagnosis. Clin Sci (Lond) 2005;109:365–79. PubMed

Wong ML, Medrano JF. Real-time PCR for mRNA quantitation. Biotechniques. 2005;39:75–85. PubMed

Bustin SA, Nolan T. Pitfalls of quantitative real-time reverse-transcription polymerase chain reaction. J Biomol Tech. 2004;15:155–66. PubMed PMC

Hsiao LL, Dangond F, Yoshida T, Hong R, Jensen RV, Misra J, Dillon W, Lee KF, Clark KE, Haverty P, Weng Z, Mutter GL, Frosch MP, Macdonald ME, Milford EL, Crum CP, Bueno R, Pratt RE, Mahadevappa M, Warrington JA, Stephanopoulos G, Stephanopoulos G, Gullans SR. A compendium of gene expression in normal human tissues. Physiol Genomics. 2001;7:97–104. PubMed

Warrington JA, Nair A, Mahadevappa M, Tsyganskaya M. Comparison of human adult and fetal expression and identification of 535 housekeeping/maintenance genes. Physiol Genomics. 2000;2:143–7. PubMed

Ishii T, Wallace AM, Zhang X, Gosselink J, Abboud RT, English JC, Pare PD, Sandford AJ. Stability of housekeeping genes in alveolar macrophages from COPD patients. Eur Respir J. 2006;27:300–6. doi: 10.1183/09031936.06.00090405. PubMed DOI

Liu DW, Chen ST, Liu HP. Choice of endogenous control for gene expression in nonsmall cell lung cancer. Eur Respir J. 2005;26:1002–8. doi: 10.1183/09031936.05.00050205. PubMed DOI

Bloor CA, Knight RA, Kedia RK, Spiteri MA, Allen JT. Differential mRNA expression of insulin-like growth factor-1 splice variants in patients with idiopathic pulmonary fibrosis and pulmonary sarcoidosis. Am J Respir Crit Care Med. 2001;164:265–72. PubMed

Muhlebach MS, Reed W, Noah TL. Quantitative cytokine gene expression in CF airway. Pediatr Pulmonol. 2004;37:393–9. doi: 10.1002/ppul.20010. PubMed DOI

Zhao L, Leung JK, Yamamoto H, Goswami S, Kheradmand F, Vu TH. Identification of P311 as a potential gene regulating alveolar generation. Am J Respir Cell Mol Biol. 2006;35:48–54. doi: 10.1165/rcmb.2005-0475OC. PubMed DOI PMC

Ye SQ, Simon BA, Maloney JP, Zambelli-Weiner A, Gao L, Grant A, Easley RB, McVerry BJ, Tuder RM, Standiford T, Brower RG, Barnes KC, Garcia JG. Pre-B-cell colony-enhancing factor as a potential novel biomarker in acute lung injury. Am J Respir Crit Care Med. 2005;171:361–70. doi: 10.1164/rccm.200404-563OC. PubMed DOI

Choi ES, Pierce EM, Jakubzick C, Carpenter KJ, Kunkel SL, Evanoff H, Martinez FJ, Flaherty KR, Moore BB, Toews GB, Colby TV, Kazerooni EA, Gross BH, Travis WD, Hogaboam CM. Focal interstitial CC chemokine receptor 7 (CCR7) expression in idiopathic interstitial pneumonia. J Clin Pathol. 2006;59:28–39. doi: 10.1136/jcp.2005.026872. PubMed DOI PMC

Glare EM, Divjak M, Bailey MJ, Walters EH. beta-Actin and GAPDH housekeeping gene expression in asthmatic airways is variable and not suitable for normalising mRNA levels. Thorax. 2002;57:765–70. doi: 10.1136/thorax.57.9.765. PubMed DOI PMC

Chambers RC. Gene expression profiling: good housekeeping and a clean message. Thorax. 2002;57:754–6. doi: 10.1136/thorax.57.9.754. PubMed DOI PMC

Zhang X, Ding L, Sandford AJ. Selection of reference genes for gene expression studies in human neutrophils by real-time PCR. BMC Mol Biol. 2005;6:4–11. doi: 10.1186/1471-2199-6-4. PubMed DOI PMC

Petrek M, Kolek V. T-lymphocyte subpopulations in bronchoalveolar lavage in pulmonary sarcoidosis and other interstitial pulmonary diseases. Cas Lek Cesk. 1993;132:365–368. [in Czech] PubMed

Meyer KC. The role of bronchoalveolar lavage in interstitial lung disease. Clin Chest Med. 2004;25:637–49. doi: 10.1016/j.ccm.2004.08.001. PubMed DOI

Petrek M, Kolek V, Szotkowska J, du Bois RM. CC and C chemokine expression in pulmonary sarcoidosis. Eur Respir J. 2002;20:1206–12. doi: 10.1183/09031936.02.00289902. PubMed DOI

Wellek S. Testing Statistical Hypotheses of Equivalence. London: Chapman and Hall/CRC Press; 2003.

Haller F, Kulle B, Schwager S, Gunawan B, von Heydebreck A, Sultmann H, Fuzesi L. Equivalence test in quantitative reverse transcription polymerase chain reaction: confirmation of reference genes suitable for normalization. Anal Biochem. 2004;335:1–9. doi: 10.1016/j.ab.2004.08.024. PubMed DOI

Pfaffl MW, Tichopad A, Prgomet C, Neuvians TP. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper – Excel-based tool using pair-wise correlations. Biotechnol Lett. 2004;26:509–15. doi: 10.1023/B:BILE.0000019559.84305.47. PubMed DOI

Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 2002;3:RESEARCH0034. doi: 10.1186/gb-2002-3-7-research0034. PubMed DOI PMC

Andersen CL, Jensen JL, Orntoft 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;64:5245–50. doi: 10.1158/0008-5472.CAN-04-0496. PubMed DOI

Freeman WM, Walker SJ, Vrana KE. Quantitative RTPCR: pitfalls and potential. Biotechniques. 1999;26:112–5. PubMed

Trapnell BC. Quantitative evaluation of gene expression in freshly isolated human respiratory epithelial cells. Am J Physiol. 1993;264:L199–212. PubMed

Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 2001;29:e45. doi: 10.1093/nar/29.9.e45. PubMed DOI PMC

Suzuki T, Higgins PJ, Crawford DR. Control selection for RNA quantitation. Biotechniques. 2000;29:332–7. PubMed

Karge WH, Schaefer EJ, Ordovas JM. Quantification of mRNA by polymerase chain reaction (PCR) using an internal standard and a nonradioactive detection method. Methods Mol Biol. 1998;110:43–61. PubMed

Fleige S, Pfaffl MW. RNA integrity and the effect on the real-time qRT-PCR performance. Mol Aspects Med. 2006;27:126–39. doi: 10.1016/j.mam.2005.12.003. PubMed DOI

Antonov J, Goldstein DR, Oberli A, Baltzer A, Pirotta M, Fleischmann A, Altermatt HJ, Jaggi R. Reliable gene expression measurements from degraded RNA by quantitative real-time PCR depend on short amplicons and a proper normalization. Lab Invest. 2005;85:1040–50. doi: 10.1038/labinvest.3700303. PubMed DOI

Silver N, Best S, Jiang J, Thein SL. Selection of housekeeping genes for gene expression studies in human reticulocytes using real-time PCR. BMC Mol Biol. 2006;7:33–41. doi: 10.1186/1471-2199-7-33. PubMed DOI PMC

de Kok JB, Roelofs RW, Giesendorf BA, Pennings JL, Waas ET, Feuth T, Swinkels DW, Span PN. Normalization of gene expression measurements in tumor tissues: comparison of 13 endogenous control genes. Lab Invest. 2005;85:154–9. PubMed

Robinson TL, Sutherland IA, Sutherland J. Validation of candidate bovine reference genes for use with real-time PCR. Vet Immunol Immunopathol. 2007;115:160–5. doi: 10.1016/j.vetimm.2006.09.012. PubMed DOI

Jesnowski R, Backhaus C, Ringel J, Löhr M. Ribosomal highly basic 23-kDa protein as a reliable standard for gene expression analysis. Pancreatology. 2002;2:421–4. doi: 10.1159/000065091. PubMed DOI

Gabrielsson BG, Olofsson LE, Sjögren A, Jernås M, Elander A, Lönn M, Rudemo M, Carlsson LM. Evaluation of reference genes for studies of gene expression in human adipose tissue. Obes Res. 2005;13:649–52. doi: 10.1038/oby.2005.72. PubMed DOI

Abruzzo LV, Lee KY, Fuller A, Silverman A, Keating MJ, Medeiros LJ, Coombes KR. Validation of oligonucleotide microarray data using microfluidic low-density arrays: a new statistical method to normalize real-time RT-PCR data. Biotechniques. 2005;38:785–92. PubMed

Coulson DT, Brockbank S, Quinn JG, Murphy S, Ravid R, Irvine GB, Johnston JA. Identification of valid reference genes for the normalization of RT qPCR gene expression data in human brain tissue. BMC Mol Biol. 2008;9:46. doi: 10.1186/1471-2199-9-46. PubMed DOI PMC

Pombo-Suarez M, Calaza M, Gomez-Reino JJ, Gonzalez A. Reference genes for normalization of gene expression studies in human osteoarthritic articular cartilage. BMC Mol Biol. 2008;9:17. doi: 10.1186/1471-2199-9-17. PubMed DOI PMC

Lyng MB, Laenkholm AV, Pallisgaard N, Ditzel HJ. Identification of genes for normalization of real-time RT-PCR data in breast carcinomas. BMC Cancer. 2008;8:20. doi: 10.1186/1471-2407-8-20. PubMed DOI PMC

Dydensborg AB, Herring E, Auclair J, Tremblay E, Beaulieu JF. Normalizing genes for quantitative RT-PCR in differentiating human intestinal epithelial cells and adenocarcinomas of the colon. Am J Physiol Gastrointest Liver Physiol. 2006;290:G1067–74. doi: 10.1152/ajpgi.00234.2005. PubMed DOI

Ohl F, Jung M, Radonic A, Sachs M, Loening SA, Jung K. Identification and validation of suitable endogenous reference genes for gene expression studies of human bladder cancer. J Urol. 2006;175:1915–20. doi: 10.1016/S0022-5347(05)00919-5. PubMed DOI

Robinson BW, McLemore TL, Crystal RG. Gamma interferon is spontaneously released by alveolar macrophages and lung T lymphocytes in patients with pulmonary sarcoidosis. J Clin Invest. 1985;75:1488–95. doi: 10.1172/JCI111852. PubMed DOI PMC

Shigehara K, Shijubo N, Ohmichi M, Kon S, Shibuya Y, Takahashi R, Morita-Ichimura S, Tatsuno T, Hiraga Y, Abe S, Sato N. Enhanced mRNA expression of Th1 cytokines and IL-12 in active pulmonary sarcoidosis. Sarcoidosis Vasc Diffuse Lung Dis. 2000;17:151–7. PubMed

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