Technical Aspects of Quantitative Competitive PCR

Zimmermann, Klaus F.; Mannhalter, Josef W.

doi:10.2144/96212rv01

Cited by 258 publications

(162 citation statements)

References 4 publications

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“…2A), any potential variations in cDNA synthesis and PCR reaction equally affected standard and wild-type amplicons. 27 Cross-validation of this RT-PCR technique with quantitative Northern blotting has demonstrated that the accuracy of both methods is similar. 13 As shown in (1) 300 ng of total RNA (containing the wildtype mRNAs), defined copy numbers of RNA standards for each of the 8 transporters and GAPDH, and specific downstream primers were used for cDNA synthesis.…”

Section: Determination Of Mrna Copy Numbersmentioning

confidence: 99%

Hepatobiliary transporter expression in percutaneous liver biopsies of patients with cholestatic liver diseases

et al. 2001

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Reduced hepatobiliary transporter expression could explain impaired hepatic uptake and excretion of bile salts and other biliary constituents resulting in cholestasis and jaundice. Because little is known about alterations of hepatobiliary transport systems in human cholestatic liver diseases, it was the aim of this study to investigate such potential changes. Hepatic mRNA levels in hepatobiliary transport systems for bile salts (NTCP, BSEP), organic anions (OATP2, MRP2, MRP3), organic cations (MDR1), phospholipids (MDR3), and aminophospholipids (FIC1) were determined in 37 human liver biopsies and control livers by competitive reverse-transcription polymerase chain reaction (RT-PCR). Transporter tissue distribution was investigated by immunofluorescence microscopy. In patients with inflammation-induced icteric cholestasis (mainly cholestatic alcoholic hepatitis), mRNA levels of NTCP, OATP2, and BSEP were reduced by 41% (P < .001), 49% (P < .005), and 34% (P < .05) compared with controls, respectively. In addition, NTCP and BSEP immunostaining was reduced. MRP2 mRNA levels remained unchanged, but canalicular immunolabeling for MRP2 was also decreased.

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Section: Determination Of Mrna Copy Numbersmentioning

confidence: 99%

Hepatobiliary transporter expression in percutaneous liver biopsies of patients with cholestatic liver diseases

et al. 2001

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“…28,29 Quantitative PCR analysis has been achieved by competitive PCR. 30 Expert laboratories showed that this technique enables accurate prediction of relapse suggesting that such analysis could be used for adapting treatment in BCR-ABLpositive CML 31,32 or in AML patients with CBFB-MYH11 or AML1-ETO transcripts. 33,34 However, this competitive PCR is labor intensive and time consuming which prohibits both standardization and large-scale multicenter analysis.…”

Section: Introductionmentioning

confidence: 99%

Standardization and quality control studies of ‘real-time’ quantitative reverse transcriptase polymerase chain reaction of fusion gene transcripts for residual disease detection in leukemia – A Europe Against Cancer Program

et al. 2003

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Detection of minimal residual disease (MRD) has proven to provide independent prognostic information for treatment stratification in several types of leukemias such as childhood acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML) and acute promyelocytc leukemia. This report focuses on the accurate quantitative measurement of fusion gene (FG) transcripts as can be applied in 35-45% of ALL and acute myeloid leukemia, and in more than 90% of CML. A total of 26 European university laboratories from 10 countries have collaborated to establish a standardized protocol for TaqManbased real-time quantitative PCR (RQ-PCR) analysis of the main leukemia-associated FGs within the Europe Against Cancer (EAC) program. Four phases were scheduled: (1) training, (2) optimization, (3) sensitivity testing and (4) patient sample testing. During our program, three quality control rounds on a large series of coded RNA samples were performed including a balanced randomized assay, which enabled final validation of the EAC primer and probe sets. The expression level of the nine major FG transcripts in a large series of stored diagnostic leukemia samples (n ¼ 278) was evaluated. After normalization, no statistically significant difference in expression level was observed between bone marrow and peripheral blood on paired samples at diagnosis. However, RQ-PCR revealed marked differences in FG expression between transcripts in leukemic Correspondence: Professor J

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“…The amplifications were performed in 50 µl volumes of 37µl H 2 O, 5 µl 1.5 mM dNTPs (mix), 5 µl 10X buffer (100 mM Tris-HCl, ph 8.3; 500 mM KCl; 15 mM MgCl 2 ; 0.01% gelatin), 0.5 µl 100 mM of each primer, 0.125 µl Taq Gold Polymerase (Applied Biosystems) and 2 µl template. To estimate the number of starting molecules in the template a competitive PCR was carried out (Zimmermann & Mannhalter 1996). In competitive PCR, a known amount of a DNA fragment (competitor) is added to the sample.…”

Section: Methodsmentioning

confidence: 99%

Techniques of DNA-studies on prehispanic ectoparasites (Pulex sp., Pulicidae, Siphonaptera) from animal mummies of the Chiribaya Culture, Southern Peru

Dittmar

Mamat

Whiting

et al. 2003

Mem. Inst. Oswaldo Cruz

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Technical Aspects of Quantitative Competitive PCR

Cited by 258 publications

References 4 publications

Hepatobiliary transporter expression in percutaneous liver biopsies of patients with cholestatic liver diseases

Hepatobiliary transporter expression in percutaneous liver biopsies of patients with cholestatic liver diseases

Standardization and quality control studies of ‘real-time’ quantitative reverse transcriptase polymerase chain reaction of fusion gene transcripts for residual disease detection in leukemia – A Europe Against Cancer Program

Techniques of DNA-studies on prehispanic ectoparasites (Pulex sp., Pulicidae, Siphonaptera) from animal mummies of the Chiribaya Culture, Southern Peru

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