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Quantitative competitive reverse transcription polymerase chain reaction is not a useful method for quantification of
Heather B Jaspan1, H Richard Gaumer, Robert F Garry
1Interdisciplinary Program in Molecular and Cellular Biology Tulane University School of Medicine, New Orleans, LA 70112, USA. hjaspan@fhcrc.org
Background:
A polymerase chain reaction (PCR)-based method for quantitating CD4 and CD8 mRNA could provide a means of assessing immune status of AIDS patients and other immunologically compromised persons without requiring large blood draws, and could be exquisitely sensitive. Such a method would also be useful in assessing the immune status of patients retrospectively.
Results:
Quantitative competitive reverse transcription PCR (QC-RT-PCR) assays were developed for measurement of CD4 and CD8 mRNA. Samples were obtained from HIV-positive and negative patients whose CD4 and CD8 counts had been determined via Flow Cytometry. The quantity of CD4 (n = 13) and CD8 (n = 28) mRNA standardized according to GAPDH mRNA quantities, all determined by QC-RT-PCR, were compared to cell number as determined by flow cytometry. There was no correlation between CD4 and CD8 cell counts and mRNA levels of CD4 and CD8 as determined by QC-RT-PCR. There is no correlation between CD4 and CD8 mRNA levels and the number of cells expressing these proteins on their surface.
Conclusion:
QC-RT-PCR, and related methodologies are not useful substitutes for assessment of CD4 and CD8 cell numbers in HIV-infected persons.