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

Nguewa PA, Agorreta J, Blanco D, Lozano MD, Gomez-Roman J, Sanchez BA, Valles I, Pajares MJ, Pio R, Rodriguez MJ, Montuenga LM, Calvo A - BMC Mol. Biol. (2008)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Oncology, Center for Applied Medical Research (CIMA), University of Navarra, Avda, Pio XII, 55, 31008 Pamplona, Spain. panguewa@unav.es

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 /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.

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.

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Related in: MedlinePlus

Variations of GAPDH and HPRT1 expression levels. Mean ± SEM of absolute values of ΔCt (/ΔCt/ = /Ct Normal-Ct Tumor/) of the two commonly used reference genes (GAPDH and HPRT1) in paired lung clinical samples (Set A).
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Figure 2: Variations of GAPDH and HPRT1 expression levels. Mean ± SEM of absolute values of ΔCt (/ΔCt/ = /Ct Normal-Ct Tumor/) of the two commonly used reference genes (GAPDH and HPRT1) in paired lung clinical samples (Set A).

Mentions: GAPDH and HPRT1 have been recommended as suitable reference genes for lung cancer research [21]. Thus, we first evaluated expression levels of these genes in the Set A samples. The analysis revealed that gene mRNA levels were significantly higher in tumors than in non-malignant tissues for both genes: GAPDH (p = 0.0001) and HPRT1 (p = 0.00003). Ct variations (ΔCt = Ct Normal-Ct Tumor) were calculated for each gene (Supplemental Table 2, Additional file 1). Ideally, a good reference gene should have /ΔCt/ values close to zero with low SEM. However, as shown in Figure 2, neither GAPDH (/ΔCt/ = 2.27 ± 0.31) nor HPRT1 (/ΔCt/ = 1.81 ± 0.26) showed this pattern.


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

Nguewa PA, Agorreta J, Blanco D, Lozano MD, Gomez-Roman J, Sanchez BA, Valles I, Pajares MJ, Pio R, Rodriguez MJ, Montuenga LM, Calvo A - BMC Mol. Biol. (2008)

Variations of GAPDH and HPRT1 expression levels. Mean ± SEM of absolute values of ΔCt (/ΔCt/ = /Ct Normal-Ct Tumor/) of the two commonly used reference genes (GAPDH and HPRT1) in paired lung clinical samples (Set A).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2612021&req=5

Figure 2: Variations of GAPDH and HPRT1 expression levels. Mean ± SEM of absolute values of ΔCt (/ΔCt/ = /Ct Normal-Ct Tumor/) of the two commonly used reference genes (GAPDH and HPRT1) in paired lung clinical samples (Set A).
Mentions: GAPDH and HPRT1 have been recommended as suitable reference genes for lung cancer research [21]. Thus, we first evaluated expression levels of these genes in the Set A samples. The analysis revealed that gene mRNA levels were significantly higher in tumors than in non-malignant tissues for both genes: GAPDH (p = 0.0001) and HPRT1 (p = 0.00003). Ct variations (ΔCt = Ct Normal-Ct Tumor) were calculated for each gene (Supplemental Table 2, Additional file 1). Ideally, a good reference gene should have /ΔCt/ values close to zero with low SEM. However, as shown in Figure 2, neither GAPDH (/ΔCt/ = 2.27 ± 0.31) nor HPRT1 (/ΔCt/ = 1.81 ± 0.26) showed this pattern.

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Oncology, Center for Applied Medical Research (CIMA), University of Navarra, Avda, Pio XII, 55, 31008 Pamplona, Spain. panguewa@unav.es

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 /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.

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.

Show MeSH
Related in: MedlinePlus