Nonradioisotopic Quantitative RT-PCR to Detect Changes in mRNA Levels during Early Mouse Embryo Development

Yokoi, Hiromichi; Natsuyama, S.; Iwai, Mieko; Noda, Y.; Mori, Tomohiro; Mori, Kazuhiro; Fujita, Katsuhide; Nakayama, Hiroki; Fujita, Jun

doi:10.1006/bbrc.1993.2112

Cited by 102 publications

(42 citation statements)

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“…The realtime PCR methods we have used are based on a kinetic analysis of the amplification curve. [35][36][37][38][39] At present, real-time PCR system is seemed to be the most sensitive and rapid system for the quantitation of DNA and RNA. 40 Real-time PCR systems were improved by probe-based, rather than intercalator-based PCR product detection.…”

Section: Discussionmentioning

confidence: 99%

O⁶‐methylguanine‐DNA methyltranspherase gene expression in gliomas by means of real‐time quantitative RT‐PCR and clinical response to nitrosoureas

Tanaka

Kobayashi

Utsuki

et al. 2002

Intl Journal of Cancer

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Key words: MGMT; real-time RT-PCR; ACNU; gliomaNitrosoureas are alkylating agents that cause cell death by binding to DNA. Among nitrosoureas, 1-(4-amino-2-methyl-5-pyrimidynyl)methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU) is widely used in Japan to treat gliomas because of its ability to permeate the blood-brain-barrier and excellent clinical effects in combination with radiation and interferon (IFN)-␤. [1][2][3] Drug-resistance genes are some of the most important elements of tumors themselves in determining drug-resistance, 4 and O 6 -methylguanine-DNA methyltransferase (MGMT) is a drug-resistance gene for nitrosoureas. 5,6 The cross-linking of doublestranded DNA by alkylating agents is inhibited by the cellular DNA-repair protein MGMT. MGMT rapidly reverses alkylation at the O 6 position of guanine, thereby averting lethal cross-linking. 7 This is the mechanism by which MGMT induces resistance to alkylating agents such as ACNU.Protocol studies for malignant gliomas have not provided encouraging therapeutic results because of the heterogeneity and various drug-sensitivities of the tumors. 8,9 Individualization of glioma therapy is recommended. We carried out a new adjuvant therapy that was individualized based on the results of the reverse transcription-polymerase chain reaction (RT-PCR) for MGMT to treat malignant gliomas. 2 Our preliminary individual adjuvant therapies (IAT) based on RT-PCR results regarding MGMT expression seemed to be more effective than conventional therapies for malignant gliomas.The level of MGMT varies widely according to the type of tumor, and even varies among tumors of the same histological classification. 5,10 Approximately 30 -50% of gliomas lack MGMT. 11 The fact that more than 50% of gliomas express MGMT limits the indications for ACNU. In our preliminary IAT, platinum (Pt)-compounds were used instead of ACNU even though Ptcompounds had not been accepted as being effective in gliomas. 12,13 To ensure IAT for gliomas, quantitative evaluation is needed for a higher initial response and prolongation of survival.We carried out the present study to determine more appropriate indications for the use of ACNU in gliomas. Real-time quantitative RT-PCR was used to re-evaluate the treated gliomas. MATERIAL AND METHODS Real-time quantitative RT-PCRIn 100 frozen sample of neuroepithelial tumors (19 low-grade neuroepithelial tumors, 28 Grade III gliomas, 41 glioblastomas, and 12 medulloblastomas), MGMT was quantitated by real-time quantitative RT-PCR using SYBR Green dye. RT-PCR was basically carried out as described previously. 14 -16 Briefly, total RNA was extracted from frozen tissue specimens weighing about 1 g, which had been homogenized using a glass Teflon homogenizer, via the guanidinium thiocyanate-phenol-chloroform single-step extraction method with Isogen (Nippon Gene, Toyama, Japan). 17 After ethanol precipitation, about 100 g of total RNA was extracted. Forty microliters of complementary deoxyribonucleic acid (cDNA) solution was synthesized from 2 g of total RNA wi...

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Section: Discussionmentioning

confidence: 99%

O⁶‐methylguanine‐DNA methyltranspherase gene expression in gliomas by means of real‐time quantitative RT‐PCR and clinical response to nitrosoureas

Tanaka

Kobayashi

Utsuki

et al. 2002

Intl Journal of Cancer

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“…The nonradioisotopic quantitative RT-PCR method was used to determine mRNA levels in different rose organs (Yokoi et al, 1993). mRNA was prepared from various floral organs and leaves as described previously (Wu et al, 2003).…”

Section: Quantitative Analysis Of Mrna Transcriptsmentioning

confidence: 99%

The Key Role of Phloroglucinol O-Methyltransferase in the Biosynthesis of Rosa chinensis Volatile 1,3,5-Trimethoxybenzene

Watanabe

Mita

et al. 2004

Plant Physiology

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1,3,5-Trimethoxybenzene is a key component of the Chinese rose odor. This compound is synthesized in three successive methylation steps from phloroglucinol, the initial precursor. A novel, to our knowledge, phloroglucinol O-methyltransferase (POMT) characterized here methylates the first step to produce the intermediate 3,5-dihydroxyanisole, while two previously described orcinol O-methyltransferases catalyze the subsequent steps. We isolated POMT from rose petals and determined partial amino acid sequences of the purified enzyme. The full-length POMT cDNA was isolated and expressed in Escherichia coli. Both the native and recombinant POMT exhibited substrate specificity for phloroglucinol. POMT was expressed specifically in floral organs, in accordance with its role as a key enzyme in the synthesis of rose floral scent compounds.

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“…The PCR products were examined by 1.5% agarose gel electrophoresis with ethidium bromide staining. To determine the relative levels of gene expression, semiquantitative analysis was performed by the method reported by Nakayama et al (1) and Yokoi et al (2). The optical density of each band was measured, and the background intensity was subtracted from the band density using image-analysis software (GDS-8000, UVP, USA).…”

Section: Detection Of Valdecoxib-induced Apoptosismentioning

confidence: 99%

The regulatory effect of the p38 signaling pathway on valdecoxib-induced apoptosis of the Eca109 cell line

Liu

Zhang²,

Guo³

et al. 2009

Oncol Rep

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Abstract. Valdecoxib is a second generation selective COX-2 inhibitor that can induce cell apoptosis in a variety of cell types, but its precise regulatory mechanism is unknown. Apoptosis of Eca109 cells and p38 mRNA expression were investigted. The expression of p-p38MAPK, Fas and FasL proteins were detected by immunohistochemical staining and FCM. Valdecoxib increased the apoptosis rate of Eca109 cells. Fas and FasL protein expression was upregulated in the valdecoxib groups, while SB203580 partly inhibited the valdecoxib-induced overexpression. Valdecoxib increased p38MAPK expression, while SB203580 inhibited the overexpression of this protein and the apoptosis rate decreased. The expression of Fas, FasL and p38MAPK protein were positively correlated with the apoptotic rate. In conclusion, valdecoxib activates the p38MAPK pathway, thus up-regulating expression of the Fas and FasL proteins, which may be one of the mechanisms through which valdecoxib induces apoptosis.

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Nonradioisotopic Quantitative RT-PCR to Detect Changes in mRNA Levels during Early Mouse Embryo Development

Cited by 102 publications

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O⁶‐methylguanine‐DNA methyltranspherase gene expression in gliomas by means of real‐time quantitative RT‐PCR and clinical response to nitrosoureas

O⁶‐methylguanine‐DNA methyltranspherase gene expression in gliomas by means of real‐time quantitative RT‐PCR and clinical response to nitrosoureas

The Key Role of Phloroglucinol O-Methyltransferase in the Biosynthesis of Rosa chinensis Volatile 1,3,5-Trimethoxybenzene

The regulatory effect of the p38 signaling pathway on valdecoxib-induced apoptosis of the Eca109 cell line

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Nonradioisotopic Quantitative RT-PCR to Detect Changes in mRNA Levels during Early Mouse Embryo Development

Cited by 102 publications

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O6‐methylguanine‐DNA methyltranspherase gene expression in gliomas by means of real‐time quantitative RT‐PCR and clinical response to nitrosoureas

O6‐methylguanine‐DNA methyltranspherase gene expression in gliomas by means of real‐time quantitative RT‐PCR and clinical response to nitrosoureas

The Key Role of Phloroglucinol O-Methyltransferase in the Biosynthesis of Rosa chinensis Volatile 1,3,5-Trimethoxybenzene

The regulatory effect of the p38 signaling pathway on valdecoxib-induced apoptosis of the Eca109 cell line

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O⁶‐methylguanine‐DNA methyltranspherase gene expression in gliomas by means of real‐time quantitative RT‐PCR and clinical response to nitrosoureas

O⁶‐methylguanine‐DNA methyltranspherase gene expression in gliomas by means of real‐time quantitative RT‐PCR and clinical response to nitrosoureas