Quantifying Aotus monkey cytokines by real-time quantitative RT-PCR.
Aotus spp. monkeys are considered the ideal model for studying the progress of malarial infection and the immune response it elicits. We describe the use of a recently developed technique, real-time quantitative RT-PCR, to quantify several Aotus monkey cytokine mRNAs involved in Th1/Th2 responses (IL-4, IL-10, TNF-beta and IFN-gamma). Specific primers were designed for each cytokine and standard curves were constructed using serial dilutions of pDNA containing each target sequence. Results were normalized to GAPDH housekeeping gene expression levels. Standard curves showed high correlation coefficients and were linear over a wide range of copy numbers. Quantification of Aotus samples showed little intra- and inter-experiment variation, thus, the technique has proven to be highly reproducible and sensitive allowing us to detect as little as 25 copies/microl of target DNA. This technique will allow studying Th1 and Th2 cytokine patterns elicited in response to infection for prospectively evaluating the efficacy of malarial vaccines.
Pico de Coaña
Yago
Y
Fundación Instituto de Inmunología de Colombia, Bogotá, Colombia.
Barrero
Carlos
C
Cajiao
Isabela
I
Mosquera
Catalina
C
Patarroyo
Manuel Elkin
ME
Patarroyo
Manuel Alfonso
MA
D000818:Animals; D001029:Aotus trivirgatus; D001483:Base Sequence; D016207:Cytokines / Q000032:analysis* / Q000235:genetics; D017931:DNA Primers; D012333:RNA, Messenger / Q000235:genetics; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D016207:Cytokines; D017931:DNA Primers; D012333:RNA, Messenger