Research article

Identification of Importin 8 (IPO8) as the most accurate reference gene for the clinicopathological analysis of lung specimens

Paul A Nguewa¹ , Jackeline Agorreta¹ , David Blanco¹ , Maria Dolores Lozano² , Javier Gomez-Roman³ , Blas A Sanchez⁴ , Iñaki Valles¹ , Maria J Pajares¹ , Ruben Pio¹ , Maria Jose Rodriguez⁴ , Luis M Montuenga^1* and Alfonso Calvo^1*

¹  Division of Oncology, Center for Applied Medical Research (CIMA), University of Navarra, Avda, Pio XII, 55, 31008 Pamplona, Spain

²  Department of Pathology, University Hospital of Navarra, Avda, Pio XII, 36, 31008 Pamplona, Spain

³  Department of Anatomical Pathology, Marqués de Valdecilla University Hospital, Medical Faculty, University of Cantabria, Santander, Spain

⁴  Research Department, Ingenasa, Madrid, Spain

author email corresponding author email* Contributed equally

BMC Molecular Biology 2008, 9:103doi:10.1186/1471-2199-9-103

Published:	17 November 2008

Abstract

Background

The accurate normalization of differentially expressed genes in lung cancer is essential for the identification of novel therapeutic targets and biomarkers by real time RT-PCR and microarrays. Although classical "housekeeping" genes, such as GAPDH, HPRT1, and beta-actin have been widely used in the past, their accuracy as reference genes for lung tissues has not been proven.

Results

We have conducted a thorough analysis of a panel of 16 candidate reference genes for lung specimens and lung cell lines. Gene expression was measured by quantitative real time RT-PCR and expression stability was analyzed with the softwares GeNorm and NormFinder, mean of |ΔCt| (= |Ct Normal-Ct tumor|) ± SEM, and correlation coefficients among genes. Systematic comparison between candidates led us to the identification of a subset of suitable reference genes for clinical samples: IPO8, ACTB, POLR2A, 18S, and PPIA. Further analysis showed that IPO8 had a very low mean of |ΔCt| (0.70 ± 0.09), with no statistically significant differences between normal and malignant samples and with excellent expression stability.

Conclusion

Our data show that IPO8 is the most accurate reference gene for clinical lung specimens. In addition, we demonstrate that the commonly used genes GAPDH and HPRT1 are inappropriate to normalize data derived from lung biopsies, although they are suitable as reference genes for lung cell lines. We thus propose IPO8 as a novel reference gene for lung cancer samples.