RANTES and MIP-1beta mRNA expression in human peripheral blood mononuclear cells: transcript quantification using NASBA technology.
The importance of chemokines in the immune response, as well as in a range of specific disease states, is becoming increasingly apparent. The role of CC- (or beta-) chemokines and their receptors in the pathology and mechanisms of HIV-1 infection has served to intensify interest in these factors. Although the functionality of these factors resides in their protein forms, assays for the detection and quantification of these protein factors in clinical samples are not readily available. Consequently, we designed NASBA-based assays for the quantification of the mRNA encoding two members of the CC-chemokine family: RANTES and MIP-1beta. The NASBA-based assays are extremely sensitive, accurate, and reproducible across a dynamic range of at least four orders of magnitude. Inter-assay performance is comparable to intra-assay performance. We applied these methods to the analysis of normal human PBMC and PBMC from HIV-1 infected individuals. Although MIP-1beta mRNA levels are higher than RANTES levels in both populations, RANTES levels in HIV-1+ patients are higher than in normal individuals. The utility of these assays in longitudinal studies of specific subpopulations of cells, as well as their potential use in clinical diagnostics, is discussed.
Romano
J W
JW
Advanced BioScience Laboratories, Inc., 5510 Nicholson Lane, Kensington, MD 20895, USA. jromano@ablinc.com
Shurtliff
R N
RN
Lee
E M
EM
Cornelison
R
R
Than
S
S
Kaplan
M H
MH
Ginocchio
C C
CC
D001775:Blood Circulation; D054407:Chemokine CCL4; D018946:Chemokine CCL5 / Q000235:genetics*; D006801:Humans; D007963:Leukocytes, Mononuclear; D019402:Macrophage Inflammatory Proteins / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis*; D015203:Reproducibility of Results; D021161:Self-Sustained Sequence Replication / Q000379:methods*
D054407:Chemokine CCL4; D018946:Chemokine CCL5; D019402:Macrophage Inflammatory Proteins; D012333:RNA, Messenger