Evaluation of mRNA levels by the polymerase chain reaction in small cardiac tissue samples.
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.
Ito
H
H
Department of Medicine, Stanford University Medical Center, CA 94305.
Miller
S C
SC
Akimoto
H
H
Torti
S V
SV
Taylor
A
A
Billingham
M E
ME
Torti
F M
FM
D000199:Actins / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002478:Cells, Cultured; D004317:Doxorubicin / Q000633:toxicity*; D005293:Ferritins / Q000235:genetics*; D015870:Gene Expression; D008297:Male; D008969:Molecular Sequence Data; D009206:Myocardium / Q000737:chemistry*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics*; D051381:Rats; D011919:Rats, Inbred Strains; D014158:Transcription, Genetic
D000199:Actins; D012333:RNA, Messenger; D004317:Doxorubicin; D005293:Ferritins