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PSMB2 and RPL32 are suitable denominators to normalize gene expression profiles in bronchoalveolar cells.

Kriegova E, Arakelyan A, Fillerova R, Zatloukal J, Mrazek F, Navratilova Z, Kolek V, du Bois RM, Petrek M - BMC Mol. Biol. (2008)

Bottom Line: To date, no reference genes have been validated for expression studies of bronchoalveolar (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).

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Immunology, Palacky University, The Czech Republic. kriegova@yahoo.com

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.

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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.
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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.

Mentions: In order to identify the most stably expressed genes in patient subgroups by equivalence test, we applied two-fold expression change cut-off for group-wise comparisons. Genes GAPDH and PSMD2 were identified as the least stably expressed genes in BAL samples, equivalently expressed only in subgroups according gender and age (Fig. 2, data for age comparison not shown). Genes ARF1, ACTB, CANX, GAPDH, GNB2L1, G6PD, GPS1, PSMD2 were found not equivalently expressed in more than two of eight studied subgroups. Out of all studied genes, only PSMB2 and RPL32 were found equivalently expressed in all studied subgroups (Fig. 2). The comparison of results of equivalence tests for two most stable genes (PSMB2, RPL32) and two "traditional" reference genes (ACTB, GAPDH) in all subgroups is shown in Fig. 3.


PSMB2 and RPL32 are suitable denominators to normalize gene expression profiles in bronchoalveolar cells.

Kriegova E, Arakelyan A, Fillerova R, Zatloukal J, Mrazek F, Navratilova Z, Kolek V, du Bois RM, Petrek M - BMC Mol. Biol. (2008)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2529339&req=5

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.
Mentions: In order to identify the most stably expressed genes in patient subgroups by equivalence test, we applied two-fold expression change cut-off for group-wise comparisons. Genes GAPDH and PSMD2 were identified as the least stably expressed genes in BAL samples, equivalently expressed only in subgroups according gender and age (Fig. 2, data for age comparison not shown). Genes ARF1, ACTB, CANX, GAPDH, GNB2L1, G6PD, GPS1, PSMD2 were found not equivalently expressed in more than two of eight studied subgroups. Out of all studied genes, only PSMB2 and RPL32 were found equivalently expressed in all studied subgroups (Fig. 2). The comparison of results of equivalence tests for two most stable genes (PSMB2, RPL32) and two "traditional" reference genes (ACTB, GAPDH) in all subgroups is shown in Fig. 3.

Bottom Line: To date, no reference genes have been validated for expression studies of bronchoalveolar (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).

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Immunology, Palacky University, The Czech Republic. kriegova@yahoo.com

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.

Show MeSH
Related in: MedlinePlus