Identification of Importin 8 (IPO8) as the most accurate reference gene for the clinicopathological analysis of lung specimens
Paul A Nguewa (1), Jackeline Agorreta (1), David Blanco (1), Maria Dolores Lozano (2), Javier Gomez-Roman (3), Blas A Sanchez (4), Iñaki Valles (1), Maria J Pajares (1), Ruben Pio (1), Maria Jose Rodriguez (4), 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
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.
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 |?Ct| (= |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 |?Ct| (0.70 ± 0.09), with no statistically significant differences between normal and malignant samples and with excellent expression stability.
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.
CITATION BMC Mol Biol. 2008 Nov 17;9:103