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. 2008 Jul 31;9:69.
doi: 10.1186/1471-2199-9-69.

PSMB2 and RPL32 Are Suitable Denominators to Normalize Gene Expression Profiles in Bronchoalveolar Cells

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Free PMC article

PSMB2 and RPL32 Are Suitable Denominators to Normalize Gene Expression Profiles in Bronchoalveolar Cells

Eva Kriegova et al. BMC Mol Biol. .
Free PMC article

Abstract

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.

Figures

Figure 1
Figure 1
Expression levels of ten housekeeping genes in bronchoalveolar cells from the 1st cohort. Expression levels of ten HKGs in CTt values over all BAL samples (n = 71). The data are expressed as whisker box plots; the box represents the 25th–75th percentiles, the median is indicated by a bar across the box, the whiskers on each box represent the minimum and maximum values.
Figure 2
Figure 2
Equivalence test for ten housekeeping genes in the 1st cohort subgroups based on the type of lung disease, treatment, smoking status, gender, and bronchoalveolar lavage cellular composition. Differences of the means (◆) and matching symmetrical confidence intervals (-) are shown for the log2-transformed relative expression of HKGs. Y-axis represents the fold change in expression among subgroups. The deviation area [-1; 1] for a fold change ≤ 2 lies within the dashed lines. If the symmetrical confidence interval is a part of the deviation area and contains zero in them, the gene is considered to be expressed equivalently. For more details on calculation see the Additional files and for statistical methodology the references [23,24]. Mean differences were calculated as follows: Mean(interstitial diseases)-Mean(other lung diseases), Mean(treated)-Mean(untreated), Mean(males)-Mean(females), Mean(smokers)-Mean(non-smokers), and Mean(pathological BAL cell counts)-Mean(normal BAL cell counts) for macrophages, lymphocytes, neutrophils and eosinophils. Reference BAL cell counts were based on own laboratory values and correspond to Meyer [21], for more details see Methods section.
Figure 3
Figure 3
Comparison of results of equivalence tests in all studied subgroups of the 1st cohort for two most stable genes PSMB2 (the left upper part) and RPL32 (the left lower part) and two most commonly used genes ACTB (the right upper part) and GAPDH (the right lower part) in bronchoalveolar (BAL) cells. 1 – Type of lung disease; 2 – Treatment; 3 – Smoking status; 4 – Gender; 5 – BAL Macrophage count; 6 – BAL Lymphocyte count; 7 – BAL Neutrophil count; 8 – BAL Eosinophil count. For more details see the legend to Fig. 2.
Figure 4
Figure 4
Equivalence test for ten housekeeping genes in the 2nd cohort subgroups based on the presence of disease, involvement of parenchyma, presence of Löfgren's syndrome, multi-organ involvement, smoking status, gender, and bronchoalveolar lavage cellular composition. Mean differences were calculated as follows: Mean(sarcoidosis patients)-Mean(control subjects), Mean(patients with involvement of parenchyma: CXR stages II/III)-Mean(patients without involvement of parenchyma: CXR stage I), Mean(Löfgren's syndrome patients)-Mean(non-Löfgren's syndrome patients), Mean(multi-organ involvement)-Mean(involvement of lung only), Mean(smokers)-Mean(non-smokers), Mean(males)-Mean(females), and Mean(pathological BAL cell count)-Mean(normal BAL cell count) for macrophages, lymphocytes, neutrophils and eosinophils. For more details see the legend to Fig. 2.
Figure 5
Figure 5
Comparison between the relative mRNA expression (ratio target gene/reference gene) of INFG gene (the upper part) and CCL2 gene (the lower part) in unseparated bronchoalveolar cells of sarcoidosis patients (S, n = 63) and control subjects (C, n = 17) from the 2nd cohort using newly validated (PSMB2/RPL32) and "traditional" reference genes (ACTB/GAPDH) as denominators. The data are presented as a mean fold change of relative expression compared to control subjects (normalized to 1); the whiskers on each box represent the SD values. For details see Methods section Gene expression measurements by qRT-PCR. *p < 0.05.

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References

    1. 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
    1. 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. - DOI - PubMed
    1. 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. - DOI - PubMed
    1. 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. - DOI - PMC - PubMed
    1. 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

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