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Use of internally controlled reverse transcriptase-polymerase chain reaction for absolute quantitation of individual
Abstract:
Research into the relative importance of P-glycoprotein (pgp) overexpression, in comparison to other mechanisms of multidrug resistance (mdr) phenotype development, has been hampered by difficulties of measurement in clinical samples. The small size, heterogeneity and often poor quality of clinical specimens renders the quantitation of mdr mRNA species by Northern analysis or RNAse protection difficult. The reverse transcriptase-polymerase chain reaction (RT-PCR) assay has both the sensitivity and specificity to make it suitable for the analysis of mdr mRNA levels in clinical samples. It also has a significant speed advantage over Northern and RNAse protection analysis. Unfortunately the variable nature of the reactions involved have made it difficult to obtain accurate and reproducible quantitative data. To overcome this difficulty we constructed internal RNA standards to each human and rat mdr mRNA species. When included in the RT-PCR reaction these internal standards allow both comparative and absolute quantitation of individual mdr family mRNA levels.
Insights
Quantifying multidrug resistance (MDR) mRNA in clinical samples is challenging. This study introduces internal RNA standards for accurate and reproducible reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of MDR gene expression.
Area of Science:
- Biochemistry
- Molecular Biology
- Pharmacology
Background:
- Multidrug resistance (MDR) is a major challenge in cancer therapy.
- P-glycoprotein (Pgp) overexpression is a key MDR mechanism.
- Quantifying MDR at the mRNA level in clinical samples is difficult due to specimen limitations.
Purpose of the Study:
- To develop a reliable method for quantifying MDR mRNA in clinical samples.
- To overcome limitations of existing techniques like Northern analysis and RNAse protection.
- To enable accurate assessment of Pgp and other MDR-related gene expression.
Main Methods:
- Utilized reverse transcriptase-polymerase chain reaction (RT-PCR) for its sensitivity and speed.
- Developed and employed internal RNA standards for human and rat MDR mRNA species.
- Implemented RT-PCR with internal standards for comparative and absolute quantitation.
Main Results:
- The developed RT-PCR method with internal standards provides accurate and reproducible quantification of MDR mRNA.
- This approach overcomes the variability issues associated with standard RT-PCR.
- Enables precise measurement of individual MDR family mRNA levels in clinical specimens.
Conclusions:
- Internal RNA standards significantly improve the accuracy and reproducibility of RT-PCR for MDR mRNA quantification.
- This method facilitates the study of MDR mechanisms in clinical settings.
- Provides a valuable tool for assessing the role of Pgp and other MDR factors in treatment resistance.