Identification Lung specimens IPO-8 The most accurate reference gene Clinicopathological analysis
Nguewa PA, Agorreta J, Blanco D, Lozano MD, Gomez-Roman J, Sanchez BA, et al. Identification of importin 8 (IPO8) as the most accurate reference gene for the clinicopathological analysis of lung specimens. BMC Mol Biol 2008 Nov 17;9:103.
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
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 RTPCR
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.