Pancreatic GAPDH Gene Expression during Ontogeny and Acute Pancreatitis Induced by Caerulein

Calvo, Ézéquiel; Boucher, Claudia; Coulombe, Zoé; Morisset, Jean

doi:10.1006/bbrc.1997.6716

Cited by 27 publications

(17 citation statements)

References 23 publications

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“…Although GAPDH , an important gene encoding for a glycolytic pathway enzyme in carbohydrate metabolism, was frequently used as an internal control for qPCR analysis, it seems to be a good internal control only for lowly expressed genes [31]. Some studies showed GAPDH was unsuitable as an internal control due to its significant variation of expression levels between different individuals during pregnancy [32], with developmental stages [33], [34] and during the cell cycle of human cells [35], which agrees with our result when mRNA from different individuals and developmental stages were examined. For the case of 18S rRNA , this ribosomal subunit gene was previously used as internal control in the gene expression studies of rice with environmental stresses [36] and the fathead minnow fish with environmental estrogens exposure [37].…”

Section: Resultsmentioning

confidence: 99%

Validation of Reference Genes for Real-Time PCR of Reproductive System in the Black Tiger Shrimp

et al. 2012

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Gene expression of reproductive system of the black tiger shrimp (Peneaus monodon) has been widely studied to address poor maturation problem in captivity. However, a systematic evaluation of reference genes in quantitative real-time PCR (qPCR) for P. monodon reproductive organs is lacking. In this study, the stability of four potential reference genes (18s rRNA, GAPDH, β-actin, and EF1-α) was examined in the reproductive tissues in various conditions using bioinformatic tools: NormFinder and geNorm. For NormFinder, EF1-α and GAPDH ranked first and second as the most stable genes in testis groups whereas GAPDH and EF1-α were for ovaries from wild-caught broodstock and domesticated groups. EF1-α and β-actin ranked first and second for the eyestalk ablated ovaries. For geNorm, EF1-α and GAPDH had the best stability in all testis and ovaries from domesticated groups whereas EF1-α and β-actin were the best for ovaries from wild-caught and eyestalk ablated groups. Moreover, the expression levels of two well-known reproductive genes, Dmc1 and Vitellogenin, were used to validate these reference genes. When normalized to EF1-α, the expected expression patterns were obtained in all cases. Therefore, this work suggests that EF1-α is more versatile as reference genes in qPCR analysis for reproductive system in P. monodon.

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Section: Resultsmentioning

confidence: 99%

Validation of Reference Genes for Real-Time PCR of Reproductive System in the Black Tiger Shrimp

et al. 2012

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“…-Actin and GAPDH are among the most commonly used genes for this purpose. However, GAPDH expression has been shown to vary with cell cycle (7) and developmental stage (8). For -actin, several highly homologous isoforms and pseudogenes have been identified, which introduce a risk of false positivity To address the issue of choosing optimal control genes for real-time RTPCR, we first evaluated the variability in expression of -actin and GAPDH in 14 different cultured human cell lines (Figure 1).…”

Section: Resultsmentioning

confidence: 99%

Selection of appropriate control genes to assess expression of tumor antigens using real-time RT-PCR

2004

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Real-time reverse transcription PCR (RT-PCR) is a sensitive and accurate method to monitor gene expression and is often used to profile the expression of putative tumor antigens in the context of immunotherapy. However, this technique consists of several steps, including cell processing, RNA extraction, RNA storage, assessment of RNA concentration, and cDNA synthesis prior to PCR. To compensate for potential variability introduced in this procedure, the expression of housekeeping genes is commonly assessed in parallel with the expression of the gene of interest. In this study, the expression of a variety of housekeeping genes in a panel of 26 different human tumor and embryonal cell lines was assessed using real-time RT-PCR. For some control genes, the variability in expression was significant between different cell lines, despite the equalization of quantities of input RNA. The greatest variability was found for GAPDH. The lowest variability was found for beta-glucuronidase (GUS) and 18S rRNA. While real-time RT-PCR is a powerful tool for gene expression analysis, these results suggest that the choice of control genes to normalize the expression of the gene of interest is critical to the interpretation of experimental results and should be tailored to the nature of the study.

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“…However, there is overwhelming evidence suggesting that its use as an internal standard is inappropriate (Oliveira et al 1999, Thellin et al 1999. GAPDH concentrations vary significantly between different individuals , during pregnancy (Cale et al 1997), with developmental stage (Puissant et al 1994, Calvo et al 1997, during the cell cycle (Mansur et al 1993) and after the addition of the tumour promoter 12-Otetradecanoyl-phorbol-13-acetate (Spanakis 1993), dexamethasone (Oikarinen et al 1991) and carbon tetrachloride (Goldsworthy et al 1993). Insulin stimulates GAPDH transcription (Rolland et al 1995, Barroso et al 1999) through multiple insulinresponsive elements in its promoter (Alexander et al 1988, Nasrin et al 1990), at least one of which is recognised by C/EBP (Alexander-Bridges et al 1992).…”

Section: Gapdhmentioning

confidence: 99%

Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays

Bustin¹

2000

Journal of Molecular Endocrinology

3,427

2,448

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The reverse transcription polymerase chain reaction (RT-PCR) is the most sensitive method for the detection of low-abundance mRNA, often obtained from limited tissue samples. However, it is a complex technique, there are substantial problems associated with its true sensitivity, reproducibility and specificity and, as a quantitative method, it suffers from the problems inherent in PCR. The recent introduction of fluorescence-based kinetic RT-PCR procedures significantly simplifies the process of producing reproducible quantification of mRNAs and promises to overcome these limitations. Nevertheless, their successful application depends on a clear understanding of the practical problems, and careful experimental design, application and validation remain essential for accurate quantitative measurements of transcription. This review discusses the technical aspects involved, contrasts conventional and kinetic RT-PCR methods for quantitating gene expression and compares the different kinetic RT-PCR systems. It illustrates the usefulness of these assays by demonstrating the significantly different levels of transcription between individuals of the housekeeping gene family, glyceraldehyde-3-phosphate-dehydrogenase (GAPDH).

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Pancreatic GAPDH Gene Expression during Ontogeny and Acute Pancreatitis Induced by Caerulein

Cited by 27 publications

References 23 publications

Validation of Reference Genes for Real-Time PCR of Reproductive System in the Black Tiger Shrimp

Validation of Reference Genes for Real-Time PCR of Reproductive System in the Black Tiger Shrimp

Selection of appropriate control genes to assess expression of tumor antigens using real-time RT-PCR

Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays

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