ISSN:
1550-7408
Source:
Blackwell Publishing Journal Backfiles 1879-2005
Topics:
Biology
Notes:
In endemic areas, clinical manifestations of Plasmodium falciparum infection range from asymptomatic parasitaemia to life-threatening severe syndromes. Immune differences that could account for this disparity are poorly understood. Using tight criteria to classify patients into non-overlapping clinical categories, we showed that cerebral malaria and severe anaemia were distinct immunological syndromes and that a proper quantitative description of cytokine profiles in the various clinical groups is essential to the understanding of the activation of immunocompetent cells.Due to the limited size of paediatric blood samples, we chose to measure cytokine mRNA using real-time RT-PCR. We showed that RT efficiency displayed intra-and intergenic variations that have to be taken into consideration for reliable absolute RNA quantification when comparing clinical cases. We thus developed a SYBR Green I-based real-time RT-PCR method using synthetic external RNA standards specific for each gene. Absolute RNA quantification is achieved by reverse transcribing known copy numbers of this RNA standard in parallel with cellular RNA. Strictly specific primers were designed to allow the quantification of any RNA in the same thermocycling parameters for future automation. Our method gave similar results for a lower cost when compared with TaqMan, and led to reproducible and reliable absolute RNA quantification. We validated it in vitro on naïve PBMC stimulated by LPS and ex vivo on PBMC from malaria patients. This new method raises the unprecedented possibility to compare cytokine mRNA levels between different clinical groups and is a powerful tool to further study the inflammation processes associated to malaria.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1111/j.1550-7408.2005.05202003_3_2.x
Permalink
