4th International Workshop on Adverse Drug Reactions and Lipodystrophy in HIV 22-25 September 2002, San Diego, CA, USA

NEW TESTS FOR MEASUREMENT OF MITOCHONDRIAL DNA AND RNA RELATIVE TO NUCLEAR DNA BASED ON HIGH-THROUGHPUT REAL-TIME MONITORED DUPLEX AMPLIFICATION

Antiviral Therapy 2002; 7:L14 (abstract 22)

MP de Baar, E Timmermans, I Dobbelaer-Bosboom, B van Gemen and A de Ronde
Primagen, Amsterdam, The Netherlands

BACKGROUND: The emerging problems of adverse effects of highly active antiretroviral therapy (HAART) and the suspected role of mitochondrial dysfunction herein revealed the necessity of having a molecular test for the assessment of mitochondrial DNA and RNA in relation to antiviral treatment. Such an assay should measure the mitochondrial DNA and RNA copies relative to the nuclear DNA to allow a conclusion about copy numbers per cell. The test to be developed should have high-throughput properties to allow fast analysis of frozen samples from current clinical studies, and short turnaround times in sample analysis in prospective studies.

METHODS: The NASBA amplification method combined with molecular beacon probes allows real-time detection during the reaction. To further enhance the throughput and increase accuracy/precision of the test, the amplification of mitochondrial DNA and nuclear DNA were multiplexed into one tube as were the mitochondrial RNA and nuclear DNA. Methodology for analysis of mitochondrial nucleic acid further encompassed isolation with the silica-based Boom method and automated registration of the real-time result.

RESULTS: The mitochondrial DNA multiplex test was extensively analysed for assay performance using in vitro cultured cells and peripheral blood mononuclear cell (PBMC) samples of both HIV-1-positive and -negative individuals. The variability of the assay is 20%, which is more accurate relative to other amplification methods that measure mitochondrial DNA and nuclear DNA in separate reactions. The dynamic range of the mitochondrial DNA and RNA tests is two logs and can cover the range of 5 to 500 copies (suitable for PBMC), 100 to 10000 (suitable for muscle cells) or any range of two logs that fits the sample type. Our first experiment with cultured PBMC showed that as a result of the addition of nucleoside analogues to the culture, the decrease of mitochondrial RNA is delayed compared with the decrease of mitochondrial DNA.

CONCLUSIONS: The newly developed duplex tests allow high-throughput analysis of samples for mitochondrial DNA and RNA with high accuracy and precision. The first data obtained with cultured PBMCs suggests that the decrease of DNA is compensated by an increase in transcription, initially increasing the amount of mitochondrial RNA per cell and delaying the later-stage decrease of mitochondrial RNA.

Presenting author: MP de Baar

2002-09-22
22

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