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Evaluation of mRNA levels by the polymerase chain reaction in small cardiac tissue samples
1Department of Medicine, Stanford University Medical Center, CA 94305.
Abstract:
Doxurubicin is an effective and widely used chemotherapeutic agent. However, use of this drug is often limited by its cardiotoxic side effects. We have observed that an early event accompanying doxorubicin cardiomyopathy is a selective decrease in levels of muscle gene transcripts in cardiac tissue (Ito et al., Proc. Natl. Acad. Sci. USA 87: 4275-4279). Since this decrease precedes ultrastructural evidence of cardiac damage, measurements of muscle transcripts might assist in the clinical evaluation of doxorubicin cardiotoxicity. We have therefore assessed the utility of the polymerase chain reaction in the measurement of mRNA in control and doxorubicin-treated animals. These measurements were performed on small tissue samples that simulate endomyocardial biopsies. We measured cardiac alpha-actin transcripts as a fraction of ferritin heavy chain transcripts using the method described by Chelly et al. (Nature 333: 858-860, 1988). 0.5 micrograms of total RNA, an amount equivalent to that obtainable from a typical endomyocardial biopsy, was efficiently co-amplified with cardiac alpha-actin and ferritin heavy chain specific primers. The cardiac alpha-actin/ferritin heavy chain ratio calculated from the PCR results correlated well (R = 0.981) with results obtained using Northern blot analysis of 10 micrograms RNA. The correlation was maintained over a wide range of cardiac alpha-actin transcript abundance. These results show that mRNA from cardiac tissue can be estimated by the polymerase chain reaction, even from a small, endomyocardial biopsy-sized sample.
Insights
Polymerase chain reaction (PCR) can accurately measure cardiac alpha-actin mRNA in small endomyocardial biopsy samples. This technique may help evaluate doxorubicin cardiotoxicity by detecting early decreases in muscle gene transcripts.
Area of Science:
- Biochemistry
- Molecular Biology
- Cardiology
Background:
- Doxorubicin is a vital chemotherapy drug, but its use is limited by cardiotoxicity.
- Early doxorubicin cardiomyopathy involves decreased cardiac muscle gene transcripts, preceding visible damage.
- Measuring these transcripts could aid in early clinical detection of cardiotoxicity.
Purpose of the Study:
- To assess the utility of polymerase chain reaction (PCR) for quantifying mRNA in small cardiac tissue samples.
- To determine if PCR can reliably measure cardiac alpha-actin transcripts in samples simulating endomyocardial biopsies.
Main Methods:
- Utilized PCR to co-amplify cardiac alpha-actin and ferritin heavy chain specific mRNA from small RNA samples (0.5 micrograms).
- Compared PCR-derived ratios of cardiac alpha-actin/ferritin heavy chain transcripts with Northern blot analysis results.
- Employed a method adapted from Chelly et al. (Nature 333: 858-860, 1988).
Main Results:
- PCR efficiently amplified target mRNA from biopsy-sized samples.
- PCR measurements of the cardiac alpha-actin/ferritin heavy chain ratio showed high correlation (R=0.981) with Northern blot analysis.
- Accurate transcript quantification was maintained across a broad range of abundance.
Conclusions:
- PCR is a viable method for estimating mRNA levels in small cardiac tissue samples, including endomyocardial biopsies.
- This technique offers a sensitive approach for evaluating doxorubicin-induced cardiotoxicity by detecting early molecular changes.
- PCR provides a reliable and efficient alternative to traditional methods for analyzing biopsy-derived RNA.