Selection of housekeeping genes for real-time PCR in atopic human bronchial epithelial cells

¹ The UBC James Hogg iCAPTURE Center for Cardiovascular and Pulmonary Research, St. Paul's Hospital, Vancouver, Canada
² The UBC James Hogg iCAPTURE Center for Cardiovascular and Pulmonary Research, St. Paul's Hospital, Vancouver, Canada; and Dept of Medicine, University of British Columbia, Vancouver, Canada
³ Rosetta Inpharmatics, Seattle Washington, USA
⁴ The UBC James Hogg iCAPTURE Center for Cardiovascular and Pulmonary Research, St. Paul's Hospital, Vancouver, Canada; and Dept of Anesthesiology, Pharmacology, and Therapeutics, University of British Columbia, Vancouver, Canada
⁵ Dept of Respiratory Medicine, Princess Margaret Hospital for Children, Perth; School of paediatrics and Child Health, The University of Western Australia, Nedlands; Telethon Institute for Child Health Research, Subiaco, Western Australia, Australia

^* To whom correspondence should be addressed. E-mail: jhe{at}mrl.ubc.ca.

	Abstract

The stability of housekeeping genes (HKGs) is critical when performing real-time quantitative PCR. To date, the stability of common HKGs has not been systematically compared in human airway epithelial cells (AEC) in normal and atopic subjects.

Expression levels of 12 HKGs were measured in AECs from a cohort of 30 healthy, atopic without asthma or atopic asthmatic children. Gene expression stability was determined using three different VBA applets (geNorm, NormFinder and BestKeeper).

All 12 HKGs were expressed in AECs. However, hypoxanthine ribosyl transferase, transcription factor IID TATA binding protein genes were excluded from further analysis due to low expression levels. Cyclophilin A (PPIA) was ranked the most stable gene by all three methods. The expression levels of beta-actin (ACTB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were significantly different among the three groups of patients, with atopic asthmatics having the highest expression levels for both genes (p=0.042 and 0.007, respectively after Bonferroni correction).

The results suggest that PPIA is the most suitable HKG analyzed for expression studies utilizing uncultured bronchial AEC from healthy and asthmatic children and highlight the importance for researchers to validate HKGs for each experimental model.