دورية أكاديمية
Quantitative reverse transcriptase-PCR amplification of cytokine mRNA in liver biopsy specimens using a non-competitive method.
| العنوان: | Quantitative reverse transcriptase-PCR amplification of cytokine mRNA in liver biopsy specimens using a non-competitive method. |
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| المؤلفون: | Bishop GA; AW Morrow Gastroenterology and Liver Centre, Royal Prince Alfred Hospital, Camperdown, New South Wales, Australia., Rokahr KL, Lowes M, McGuinness PH, Napoli J, DeCruz DJ, Wong WY, McCaughan GW |
| المصدر: | Immunology and cell biology [Immunol Cell Biol] 1997 Apr; Vol. 75 (2), pp. 142-7. |
| نوع المنشور: | Journal Article; Research Support, Non-U.S. Gov't |
| اللغة: | English |
| بيانات الدورية: | Publisher: Wiley Country of Publication: United States NLM ID: 8706300 Publication Model: Print Cited Medium: Print ISSN: 0818-9641 (Print) Linking ISSN: 08189641 NLM ISO Abbreviation: Immunol Cell Biol Subsets: MEDLINE |
| أسماء مطبوعة: | Publication: 2018- : [Hoboken, NJ] : Wiley Original Publication: [Adelaide, South Australia] : University of Adelaide, [c1987- |
| مواضيع طبية MeSH: | Polymerase Chain Reaction/*methods , RNA, Messenger/*metabolism , Tumor Necrosis Factor-alpha/*metabolism, Biomarkers ; Biopsy ; DNA Primers ; Humans ; Liver/metabolism ; Liver/pathology ; RNA-Directed DNA Polymerase/genetics ; Reproducibility of Results ; Sensitivity and Specificity ; Tumor Necrosis Factor-alpha/genetics |
| مستخلص: | Reverse transcriptase-PCR (RT-PCR) amplification of mRNA is often the only technique able to detect expression of cytokine mRNA in small samples. The aim of this work was to investigate the utility of a non-competitive RT-PCR which used external standards to quantitate TNF-alpha mRNA in liver biopsy specimens from liver transplant patients. It involved removal of aliquots from the PCR reaction at successive cycles, followed by dot-blotting of the samples onto nylon membrane and hybridization with a radioactively-labelled internal probe. Phosphorimage analysis of the labelled membranes allowed quantitation of the relative amount of PCR product at successive cycles. Plots of log(counts) versus cycle number showed straight lines in the exponential phase of amplification. The slopes of these lines showed the efficiency of amplification, which ranged from 76 to 87% for liver biopsy samples. Estimation of liver biopsy levels of TNF-alpha in two separate PCR amplifications showed low inter-assay variability (r2 = 0.98). Comparison of two separate cDNA syntheses also showed good correlation (r2 = 0.81, P < 0.0001), although not as good as for the PCR alone. This shows that variation in efficiency of cDNA synthesis is likely to contribute as much or more to variability of the analysis as variations in PCR amplification. |
| المشرفين على المادة: | 0 (Biomarkers) 0 (DNA Primers) 0 (RNA, Messenger) 0 (Tumor Necrosis Factor-alpha) EC 2.7.7.49 (RNA-Directed DNA Polymerase) |
| تواريخ الأحداث: | Date Created: 19970401 Date Completed: 19970612 Latest Revision: 20180822 |
| رمز التحديث: | 20240628 |
| DOI: | 10.1038/icb.1997.19 |
| PMID: | 9107566 |
| قاعدة البيانات: | MEDLINE |
| تدمد: | 0818-9641 |
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| DOI: | 10.1038/icb.1997.19 |