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


CONCLUSIONS
PSMB2 and RPL32 are, therefore, suitable reference genes to normalize qRT-PCR in BAL cells in sarcoidosis, and other interstitial lung disease.
Kriegova
Eva
E
Department of Immunology, Palacky University, The Czech Republic. kriegova@yahoo.com
Arakelyan
Arsen
A
Fillerova
Regina
R
Zatloukal
Jaromir
J
Mrazek
Frantisek
F
Navratilova
Zdenka
Z
Kolek
Vitezslav
V
du Bois
Roland M
RM
Petrek
Martin
M
D000293:Adolescent; D000328:Adult; D000818:Animals; D001980:Bronchi / Q000166:cytology*; D016022:Case-Control Studies; D005260:Female; D020869:Gene Expression Profiling / Q000592:standards*; D006801:Humans; D008171:Lung Diseases / Q000235:genetics; D008297:Male; D008875:Middle Aged; D011650:Pulmonary Alveoli / Q000166:cytology*; D012333:RNA, Messenger / Q000032:analysis; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000592:standards; D012507:Sarcoidosis / Q000235:genetics*; D012907:Smoking / Q000235:genetics
D012333:RNA, Messenger