Improved quantification with validation of multiple mRNA species by polymerase chain reaction: application to human myocardial creatine kinase M and B.
OBJECTIVE
Quantitation by polymerase chain reaction (PCR) has not been validated by an independent assay and is not easily applicable to multiple samples. The aim of this study was therefore to attempt to quantify human heart creatine kinase M (CKM) and B (CKB) mRNA simultaneously and to validate the method with a quantitative S1 nuclease protection assay.


METHODS
Conditions were optimised to achieve high efficiency of reverse transcription and PCR amplification of a short target sequence using total human heart RNA and an in vitro transcribed RNA standard as a substrate. A nested radiolabelled primer was used for specific detection and quantification of the amplified DNA sequence.


RESULTS
The amplification efficiency for CKM and CKB mRNA were 0.98 (SD 0.07) and 1.11 (0.04) respectively. CKM and CKB mRNA levels were determined in 42 samples from 24 human hearts and found to be 156.7(36.2) and 19.9(5.2) amol.microgram-1 RNA, respectively. Parallel quantitative S1 nuclease protection assay yielded results of 131.2(64.2) and 9.4(5.2) amol.microgram-1 RNA. The cardiac CKB, but not CKM mRNA level, was twofold higher using the quantitative PCR method. However this discrepancy was abolished when compared to a higher stringency S1 nuclease protection assay. The cardiac CKB mRNA was 12.7% of the CKM level. This proportion remained the same from hearts with end stage cardiomyopathies of various aetiologies.


CONCLUSIONS
This validated quantitative PCR method offers advantages over the S1 nuclease protection assay in that less RNA is required, the procedure is less dependent on RNA integrity and secondary structure, and multiple RNA species can be quantified simultaneously. The results also suggest that the abundance of the CKM and CKB mRNA level are coordinately regulated in the human heart.
Ma
T S
TS
Baylor College of Medicine, Houston, Texas.
Brink
P A
PA
Perryman
B
B
Roberts
R
R
D000293:Adolescent; D000328:Adult; D000368:Aged; D001483:Base Sequence; D015152:Blotting, Northern; D009202:Cardiomyopathies / Q000175:diagnosis; D003402:Creatine Kinase / Q000235:genetics*; D017931:DNA Primers; D005260:Female; D005821:Genetic Techniques; D006801:Humans; D006973:Hypertension / Q000175:diagnosis; D007527:Isoenzymes; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D017202:Myocardial Ischemia / Q000175:diagnosis; D009206:Myocardium / Q000201:enzymology*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis*; D015203:Reproducibility of Results
D017931:DNA Primers; D007527:Isoenzymes; D012333:RNA, Messenger; D003402:Creatine Kinase