A reliability test of standard-based quantitative PCR: exogenous vs endogenous standards

Li, Ke; Chen, Z.; Yung, W. K. Alfred

doi:10.1006/mcpr.2000.0288

Cited by 100 publications

(69 citation statements)

References 30 publications

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“…Consistent with previously published findings in the muscle-specific frataxin knockout (10), we generally did not observe remnant wild-type signals by RT-PCR within the knockout tissue (Figure 1b). However, frataxin was detectable in α cells by immunohistochemistry (Figure 1d), presumably reflecting a preferential amplification of the shorter fragment, which was previously found to be typical for shortened internal standards employed in semiquantitative PCR reactions (32).…”

Section: Disruption Of the Frataxin Gene In Pancreaticsupporting

confidence: 52%

Frataxin deficiency in pancreatic islets causes diabetes due to loss of β cell mass

Ristow¹,

Mulder²,

Pomplun³

et al. 2003

J. Clin. Invest.

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Diabetes is caused by an absolute (type 1) or relative (type 2) deficiency of insulin-producing β cells. We have disrupted expression of the mitochondrial protein frataxin selectively in pancreatic β cells. Mice were born healthy but subsequently developed impaired glucose tolerance progressing to overt diabetes mellitus. These observations were explained by impairment of insulin secretion due to a loss of β cell mass in knockout animals. This phenotype was preceded by elevated levels of reactive oxygen species in knockout islets, an increased frequency of apoptosis, and a decreased number of proliferating β cells. Hence, disruption of the frataxin gene in pancreatic β cells causes diabetes following cellular growth arrest and apoptosis, paralleled by an increase in reactive oxygen species in islets. These observations might provide insight into the deterioration of β cell function observed in different subtypes of diabetes in humans

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Section: Disruption Of the Frataxin Gene In Pancreaticsupporting

confidence: 52%

Frataxin deficiency in pancreatic islets causes diabetes due to loss of β cell mass

Ristow¹,

Mulder²,

Pomplun³

et al. 2003

J. Clin. Invest.

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show abstract

“…It is especially difficult when dealing with in vivo samples and comparing gene expression patterns between different individuals. Surprisingly, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) continues to be utilised as a normaliser despite continuing reports that emphasise the problems associated with its use (Ke et al 2000, Suzuki et al 2000. It is now well documented that GAPDH mRNA levels are not constant (Zhu et al 2001), that it contributes to diverse cellular functions such as nuclear RNA export, DNA replication, DNA repair, exocytotic membrane fusion, cytoskeletal organisation and phosphotransferase activity (Sirover 1999).…”

Section: Normalisationmentioning

confidence: 99%

Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems

Bustin

2002

Journal of Molecular Endocrinology

2,232

1,674

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The fluorescence-based real-time reverse transcription PCR (RT-PCR) is widely used for the quantification of steady-state mRNA levels and is a critical tool for basic research, molecular medicine and biotechnology. Assays are easy to perform, capable of high throughput, and can combine high sensitivity with reliable specificity. The technology is evolving rapidly with the introduction of new enzymes, chemistries and instrumentation. However, while real-time RT-PCR addresses many of the difficulties inherent in conventional RT-PCR, it has become increasingly clear that it engenders new problems that require urgent attention. Therefore, in addition to providing a snapshot of the state-of-the-art in real-time RT-PCR, this review has an additional aim: it will describe and discuss critically some of the problems associated with interpreting results that are numerical and lend themselves to statistical analysis, yet whose accuracy is significantly affected by reagent and operator variability.

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“…These results are in agreement with those of Al-Bader and Al-Sarraf (2005) showing that GAPDH, albumins, actins, tubulins, Bovine cytokine gene expression 577 cyclophilins, 18S rRNA and 28S RNA, used extensively as control genes, may vary under different experimental conditions in different tissues. Although GAPDH is widely used as an internal control gene (Giulietti et al, 2001;Bustin, 2002;Bustin et al, 2005), several reports emphasize problems associated with its use (Ke et al, 2000;Suzuki et al, 2000). Al-Bader and Al-Sarraf (2005) used RT-PCR to measure mRNA levels of several genes, in order to identify those most suitable to normalize expression.…”

Section: Real-time Rt-pcrmentioning

confidence: 99%

Quantification of bovine cytokine gene expression using real-time RT-PCR methodology

et al. 2007

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T cells produce cytokines that affect host response to infection. This paper reports real-time RT-PCR conditions and validation steps for accurate quantification of Bos indicus cytokines, interleukin (IL)-2, IL-4, IL-8, IL12p-35, IL-13, tumoral necrosis factor (TNF)-α, interferon (IFN)-γ, monocyte chemoattractant proteins (MCP)-1 and MCP-2, and the glycoprotein mucin (MUC)-1 in two groups of Nelore cattle, one resistant and the other susceptible to gastrointestinal nematode infections. RPL-19 was shown to be an ideal internal control gene, since its expression was constant across treatments and presented lower variation when compared to the GAPDH gene. The optimized conditions established in the present study can be used to determine the immune response of cattle under different experimental conditions, such as viral, bacterial and parasite infections.

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A reliability test of standard-based quantitative PCR: exogenous vs endogenous standards

Cited by 100 publications

References 30 publications

Frataxin deficiency in pancreatic islets causes diabetes due to loss of β cell mass

Frataxin deficiency in pancreatic islets causes diabetes due to loss of β cell mass

Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems

Quantification of bovine cytokine gene expression using real-time RT-PCR methodology

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