Semi-Quantitative RT-PCR Method to Estimate Full-Length mRNA Levels of the Multidrug Resistance Gene
Expression levels of P-glycoprotein (P-gp), the transporter encoded by the human multidrug resistance gene (MDR1), may play an important role in drug disposition. The ability to quantitate full-length MDR1 mRNA levels may be predictive of P-gp expression and function. Therefore, a semi-quantitative...
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| Format: | Article |
| Language: | English |
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Taylor & Francis Group
2002-07-01
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| Series: | BioTechniques |
| Online Access: | https://www.future-science.com/doi/10.2144/02331dd03 |
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| author | Z. Yang E.L. Woodahl X.-Y. Wang T. Bui D.D. Shen R.J.Y. Ho |
| author_facet | Z. Yang E.L. Woodahl X.-Y. Wang T. Bui D.D. Shen R.J.Y. Ho |
| author_sort | Z. Yang |
| collection | DOAJ |
| description | Expression levels of P-glycoprotein (P-gp), the transporter encoded by the human multidrug resistance gene (MDR1), may play an important role in drug disposition. The ability to quantitate full-length MDR1 mRNA levels may be predictive of P-gp expression and function. Therefore, a semi-quantitative RT-PCR assay was developed to assess full-length MDR1 mRNA levels. Levels of full-length 3.8-kb MDR1 mRNA were estimated by comparing PCR amplification of the RNA extract with that of an internal standard, ΔMDR1. The 2.9-kb ΔMDR1 competitor RNA standard was constructed by deleting 965 bp from the interior of MDR1 mRNA. The full-length MDR1 and ΔMDR1 share identical 5′ and 3′ primer binding sequences, allowing for their simultaneous amplification in the same RT-PCR. With this approach, MDR1 mRNA levels can be sensitively and reliably estimated with a detection limit of 2000 copies. Full-length MDR1mRNA levels in various human cell lines and lymphocytes from leukemia patients varied over 100-fold, ranging from 0.3 to 36.5 × 105 copies/μg total RNA. The semi-quantitative full-length RT-PCR assay may be useful in estimating MDR1 mRNA levels to assess P-gp expression, which may be important in studying the role of P-gp in drug disposition and cancer chemotherapy efficacy. |
| format | Article |
| id | doaj-art-7bbdb021fd7b496baaae4125c912f50c |
| institution | OA Journals |
| issn | 0736-6205 1940-9818 |
| language | English |
| publishDate | 2002-07-01 |
| publisher | Taylor & Francis Group |
| record_format | Article |
| series | BioTechniques |
| spelling | doaj-art-7bbdb021fd7b496baaae4125c912f50c2025-08-20T02:26:03ZengTaylor & Francis GroupBioTechniques0736-62051940-98182002-07-0133119620310.2144/02331dd03Semi-Quantitative RT-PCR Method to Estimate Full-Length mRNA Levels of the Multidrug Resistance GeneZ. Yang0E.L. Woodahl1X.-Y. Wang2T. Bui3D.D. Shen4R.J.Y. Ho51University of Washington, Seattle, WA, USA1University of Washington, Seattle, WA, USA1University of Washington, Seattle, WA, USA1University of Washington, Seattle, WA, USA1University of Washington, Seattle, WA, USA1University of Washington, Seattle, WA, USAExpression levels of P-glycoprotein (P-gp), the transporter encoded by the human multidrug resistance gene (MDR1), may play an important role in drug disposition. The ability to quantitate full-length MDR1 mRNA levels may be predictive of P-gp expression and function. Therefore, a semi-quantitative RT-PCR assay was developed to assess full-length MDR1 mRNA levels. Levels of full-length 3.8-kb MDR1 mRNA were estimated by comparing PCR amplification of the RNA extract with that of an internal standard, ΔMDR1. The 2.9-kb ΔMDR1 competitor RNA standard was constructed by deleting 965 bp from the interior of MDR1 mRNA. The full-length MDR1 and ΔMDR1 share identical 5′ and 3′ primer binding sequences, allowing for their simultaneous amplification in the same RT-PCR. With this approach, MDR1 mRNA levels can be sensitively and reliably estimated with a detection limit of 2000 copies. Full-length MDR1mRNA levels in various human cell lines and lymphocytes from leukemia patients varied over 100-fold, ranging from 0.3 to 36.5 × 105 copies/μg total RNA. The semi-quantitative full-length RT-PCR assay may be useful in estimating MDR1 mRNA levels to assess P-gp expression, which may be important in studying the role of P-gp in drug disposition and cancer chemotherapy efficacy.https://www.future-science.com/doi/10.2144/02331dd03 |
| spellingShingle | Z. Yang E.L. Woodahl X.-Y. Wang T. Bui D.D. Shen R.J.Y. Ho Semi-Quantitative RT-PCR Method to Estimate Full-Length mRNA Levels of the Multidrug Resistance Gene BioTechniques |
| title | Semi-Quantitative RT-PCR Method to Estimate Full-Length mRNA Levels of the Multidrug Resistance Gene |
| title_full | Semi-Quantitative RT-PCR Method to Estimate Full-Length mRNA Levels of the Multidrug Resistance Gene |
| title_fullStr | Semi-Quantitative RT-PCR Method to Estimate Full-Length mRNA Levels of the Multidrug Resistance Gene |
| title_full_unstemmed | Semi-Quantitative RT-PCR Method to Estimate Full-Length mRNA Levels of the Multidrug Resistance Gene |
| title_short | Semi-Quantitative RT-PCR Method to Estimate Full-Length mRNA Levels of the Multidrug Resistance Gene |
| title_sort | semi quantitative rt pcr method to estimate full length mrna levels of the multidrug resistance gene |
| url | https://www.future-science.com/doi/10.2144/02331dd03 |
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