Ribosomal 18S RNA Prevails over Glyceraldehyde-3-Phosphate Dehydrogenase and β-Actin Genes as Internal Standard for Quantitative Comparison of mRNA Levels in Invasive and Noninvasive Human Melanoma Cell Subpopulations

doi:10.1006/abio.2001.5171

Analytical Biochemistry

Volume 295, Issue 1, 1 August 2001, Pages 17-21

https://doi.org/10.1006/abio.2001.5171 Get rights and content

Abstract

The comparison of the gene expression profiles between two subpopulations of melanoma cells (1C8 and T1C3) derived from the tumor of one patient by cDNA array revealed differences in GAPDH and β-actin gene levels. These two housekeeping genes were up-regulated in invasive T1C3 melanoma cells compared to noninvasive 1C8 cells. Since cDNA array results were not confirmed by conventional RT-PCR throughout the exponential phase of amplification, we performed duplex relative RT-PCR using ribosomal 18S RNA as internal standard including competimer technology. Statistical analyses provided significant evidence that invasive T1C3 melanoma cells exhibited a twofold higher mRNA level of both GAPDH and β-actin than noninvasive 1C8 cells. This study demonstrates that the duplex relative RT-PCR procedure including ribosomal 18S RNA as internal standard and competimer technology is precise for RNA quantification and is tailored for cDNA array validation. Our data provide molecular evidence that cellular subpopulations of the same pathological origin are highly heterogeneous and extend the concept that the selection of an appropriate internal control for comparative mRNA analysis should be adapted to each model of human cancers.

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A total of 35 DEMs were found with the criteria of P < 0.001 and absolute value of Log2 fold change > 0.58. The most significant pathways and BPs affected in poor prognosis group were associated with ubiquitination and RNA splicing. The survival analysis revealed that the aberrant expression of 22 hub genes was significantly associated with worse overall survival in OSCC. The most significant Kaplan-Meier curves were achieved from EGF, RTN4, RAN, ACTB, and CYCS genes with the Logrank test P-value of <0.01.
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¹: A “Scientifique du Contingent” from DSP/DGA/STTC (Ministry of Defense).

²: To whom correspondence should be addressed at INSERM U 346, Hôpital Edouard Herriot, Pavillon R, 69437 Lyon 03, France. Fax: + 33(0)4 72 11 02 90. E-mail: [email protected].

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Regular ArticleRibosomal 18S RNA Prevails over Glyceraldehyde-3-Phosphate Dehydrogenase and β-Actin Genes as Internal Standard for Quantitative Comparison of mRNA Levels in Invasive and Noninvasive Human Melanoma Cell Subpopulations

Abstract

Biochim. Biophys. Acta

FEBS Lett.

Biochim. Biophys. Acta

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

J. Biol. Chem.

Lancet

Anal. Biochem.

Eur. J. Cancer

Anal. Biochem.

Mechanisms of tumor progression

Cancer Res.

Oncogenes, antioncogenes, and the molecular bases of multistep carcinogenesis

Cancer Res.

Regular Article
Ribosomal 18S RNA Prevails over Glyceraldehyde-3-Phosphate Dehydrogenase and β-Actin Genes as Internal Standard for Quantitative Comparison of mRNA Levels in Invasive and Noninvasive Human Melanoma Cell Subpopulations