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

Goidin, Didier; Mamessier, Audrey; Staquet, Marie‐Jeanne; Schmitt, Daniel; Berthier‐Vergnes, Odile

doi:10.1006/abio.2001.5171

Cited by 358 publications

(233 citation statements)

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“…Glyceraldehyde-3-phosphate-dehydrogenase is an abundant glycolytic enzyme and therefore an increase in transcription is consistent with a higher demand of cellular energy associated with growth and cell differentiation. Normalisation to GAPDH was already reported as valid (Wall and Edwards 2002), however in most cases this gene proved to be inappropriate as endogenous control in quantification assays (Schmittgen and Zakrajsek 2000;Goidin et al 2001;Kim et al 2003). A maximum variability of GAPDH of 25 fold has been reported (Dheda et al 2004), and according to Bustin (2002) for most experimental conditions the use of GAPDH is inappropriate and should be discontinued.…”

Section: Discussionmentioning

confidence: 99%

“…In fact, 18S ribosomal RNA has been reported as a reliable reference gene for real-time RT-PCR in distinct experimental conditions (Schmittgen and Zakrajsek 2000;Goidin et al 2001;Kim et al 2003). However, according to Solanas et al (2001) there are some concerns regarding the use of rRNA as internal standard due to variations in rRNA expression levels, transcription by different RNA polymerases and possible imbalances in rRNA and mRNA fractions between different samples.…”

Section: Discussionmentioning

confidence: 99%

“…There have been several reports on the study of genes as internal standards for quantitative real-time PCR in animal tissues (Goidin et al 2001;Feroze-Merzoug et al 2002;Lupberger et al 2002). In plant tissues, real time RT-PCR technique is being used for quantification of gene expression (Charrier et al 2002;Mason et al 2002), and in the majority of studies 'housekeeping genes' are being employed as internal controls without an adequate validation of the stability of expression of these genes under the conditions assayed.…”

Section: Introductionmentioning

confidence: 99%

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Evaluation of control transcripts in real-time RT-PCR expression analysis during maritime pine embryogenesis

Gonçalves

Cairney

Marôco

et al. 2005

Planta

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In order to determine the suitability of reference or housekeeping genes as internal controls in real-time reverse transcriptase PCR (RT-PCR) assays for quantification of target mRNAs, we studied the levels of expression of four candidate reference genes in maritime pine by real-time RT-PCR. The expression levels obtained for glyceraldehyde-3-phosphate-dehydrogenase, 18S ribosomal RNA, eukaryotic translation initiation factor eIF4AII and ubiquitin in nine stages of embryo development revealed that none of the genes tested proved to be suitable as an internal control. Copy number quantification of the four transcripts showed an average relative variation of seven fold. We propose that the combination of a precise method for RNA quantification, internal controls for monitoring RT reaction and PCR efficiency and a robust external standard curve can guarantee a reliable absolute quantification of mRNA transcripts in real time RT-PCR. This approach may avoid the controversy in the use of housekeeping genes and may assume special significance in tissues undergoing developmental changes.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Evaluation of control transcripts in real-time RT-PCR expression analysis during maritime pine embryogenesis

Gonçalves

Cairney

Marôco

et al. 2005

Planta

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“…"Internal reference" genes are presumed to be essential for cell viability and thus constitutively expressed to maintain cellular function [6], and are widely employed as quantification controls, correlating target gene levels to that of the reference gene [7]. The expression of even the most commonly used internal controls may vary as a result of neoplastic growth, leading to inaccurate quantification [8]. In the present study, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and 18S rRNA, two of the most commonly used internal reference genes, were investigated as controls for qPCR analysis of melanoma tissue, native tissue, cultured melanoma cells, and fibroblasts.…”

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confidence: 99%

“…Several growth dependant signaling pathways have been implicated in regulating rRNA levels [13], such as the mammalian target of rapamycin (mTOR) and mitogen activated protein kinase (MAPK) pathways, both of which have been described as crucial for melanoma 14;15]. Thus, as with GAPDH, it is understandable as to why 18S rRNA is not a good choice as a control gene for melanoma and normal tissue types.A previous study found 18S rRNA to be a more reliable standard compared to GAPDH or β-actin in cultured melanoma cells [8]. However, this prior study utilized regular PCR and duplex relative PCR, both of which are based on the visualization and quantification of the PCR products by gel electrophoresis and densiometric analysis.…”

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confidence: 99%

The normalization of gene expression data in melanoma: Investigating the use of glyceraldehyde 3-phosphate dehydrogenase and 18S ribosomal RNA as internal reference genes for quantitative real-time PCR

Giricz

Lauer‐Fields

Fields

2008

Analytical Biochemistry

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We examined a panel of 26 melanoma and fibroblast samples (tissues and cultured cells) to evaluate the suitability of two commonly used housekeeping genes, GAPDH and 18S rRNA, for quantitative real-time PCR. Both genes showed significant variations within the individual cell line and tissue groups. While no overall trends were observed in the expression of the 18S rRNA, GAPDH was upregulated in melanoma tissue and cultured cells compared to the corresponding normal samples. In melanoma and fibroblast cell lines and tissues, absolute quantification appears to be more appropriate than normalizing mRNA expression via GAPDH or 18S rRNA housekeeping genes.The number of skin cancer cases has been steadily increasing with the risk of developing malignant cutaneous melanoma doubling every 10-15 years [1]. However, in 2007 the new cases of melanoma was estimated at 59,940, nearly double that of previous estimates [2]. If melanoma is detected early (localized), the five year survival rate is ∼99% [3]. However, distant stage (metastatic) melanoma has a very poor prognosis (∼15% five year survival) [3]. Thus, there is a critical requirement for accurate assessment of melanoma initiation and progression, as well as the characterization of the more malignant later stages.The most reliable prognostic factor of melanoma is still the tumor thickness [4], thus underscoring the importance of early detection and surgery. Better knowledge of the underlying molecular mechanisms of transformation and melanoma progression would possibly aid in the discovery of novel biomarkers, potential therapeutic targets, or biochemical pathways. Previous studies have shown that gene expression profiling of melanoma mRNA levels may lead to novel molecular classification methods by identifying the subtypes of disease and possibly predicting phenotypic characteristics ([5] and references cited therein). Microarray analysis can be used for the high-throughput analysis of global gene expression levels, studying hundreds or thousands of targets at once. Quantitative real-time PCR (qRT-PCR or qPCR) allows for the sensitivity to analyze a narrow range of targets or to confirm previously obtained microarray data.Currently there are two quantification methods available for qPCR, absolute and relative. In the case of absolute quantification, genomic DNA or plasmids containing cloned qPCR target *Corresponding author. Department of Chemistry & Biochemistry, Florida Atlantic University, 777 Glades Road, Boca Raton, FL 33431-0991, USA. Tel: 1-561-297-2093. Fax: 1-561-297-2759. E-mail address: fieldsg@fau.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal di...

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Using Host 28S Ribosomal RNA as a Housekeeping Gene for Quantitative Real‐Time Reverse Transcription‐PCR (qRT‐PCR) in Virus‐Infected Animal Cells

Xue

Cheng

2010

CP Microbiology

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The use of quantitative real‐time reverse transcription‐PCR (qRT‐PCR) for studying regulation of gene transcription requires an internal template‐loading control or a housekeeping gene to guarantee the validity of the data collection, analysis, and interpretation. Analysis of gene transcription in virus‐infected animal cells is problematic because virus infection often results in modified or fluctuating gene transcription patterns of conventionally used housekeeping genes, such as the glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) gene and the β‐actin gene. It has been demonstrated that the host 28S ribosomal gene can be used as a housekeeping gene in qRT‐PCR in virus‐infected insect cells. The stability of the human 28S rRNA gene transcription during the infection of HeLa cells with adenovirus has been confirmed, and this method has been extended to the use of the human 28S rRNA gene as a housekeeping gene in adenovirus‐infected HeLa cells. Step‐by‐step instructions are described for use of this control in analysis of gene transcription in both types of virus‐infected animal cells. Curr. Protoc. Microbiol. 19:1D.2.1‐1D.2.13. © 2010 by John Wiley & Sons, Inc.

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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

Cited by 358 publications

References 20 publications

Evaluation of control transcripts in real-time RT-PCR expression analysis during maritime pine embryogenesis

Evaluation of control transcripts in real-time RT-PCR expression analysis during maritime pine embryogenesis

The normalization of gene expression data in melanoma: Investigating the use of glyceraldehyde 3-phosphate dehydrogenase and 18S ribosomal RNA as internal reference genes for quantitative real-time PCR

Using Host 28S Ribosomal RNA as a Housekeeping Gene for Quantitative Real‐Time Reverse Transcription‐PCR (qRT‐PCR) in Virus‐Infected Animal Cells

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