Identification of importin 8 (IPO8) as the most accurate reference gene for the clinicopathological analysis of lung specimens.
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 |Delta 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 |Delta Ct| (0.70 +/- 0.09), with no statistically significant differences between normal and malignant samples and with excellent expression stability.


CONCLUSIONS
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.
Nguewa
Paul A
PA
Division of Oncology, Center for Applied Medical Research (CIMA), University of Navarra, Avda, Pio XII, 55, 31008 Pamplona, Spain. panguewa@unav.es
Agorreta
Jackeline
J
Blanco
David
D
Lozano
Maria Dolores
MD
Gomez-Roman
Javier
J
Sanchez
Blas A
BA
Valles
Iñaki
I
Pajares
Maria J
MJ
Pio
Ruben
R
Rodriguez
Maria Jose
MJ
Montuenga
Luis M
LM
Calvo
Alfonso
A
D002289:Carcinoma, Non-Small-Cell Lung / Q000201:enzymology / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D015972:Gene Expression Regulation, Neoplastic; D052138:Genes, Neoplasm; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) / Q000378:metabolism; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000378:metabolism; D008168:Lung / Q000378:metabolism* / Q000473:pathology*; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012984:Software; D028961:beta Karyopherins / Q000235:genetics*
C107282:IPO8 protein, human; D028961:beta Karyopherins; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating); D007041:Hypoxanthine Phosphoribosyltransferase