Quantification of canine cytokines using real time reverse transcriptase polymerase chain reaction.
BACKGROUND
Canines are the principal domestic reservoirs of visceral leishmaniasis in both the Old and New World. The development of highly sensitive and quantitative methods, such as real time reverse transcriptase polymerase chain reaction for measurement of canine cytokines has not been exploited in studies of visceral leishmaniasis.


OBJECTIVE
To standardize the relative quantification of canine IFN-gamma, IL-4, IL-10, IL-12p40 and IL-12p35 using real time reverse transcriptase polymerase chain reaction.


METHODS
RNA was isolated from PBMCs from 1 year-old foxhounds and cultured with or without Con A, LPS or Staphylococcus aureus extract. This RNA was used in one-step real time reverse transcriptase polymerase chain reaction to optimize the concentrations of the cytokine primers and probes, generate standard curves for each cytokine, confirm equivalent amplification efficiency of cytokine and normalizer (18S rRNA) RNA, and to quantify the expression of the cytokine RNA. The comparative Ct method was used to determine the relative levels of gene expression in the samples, expressed as the fold-increase relative to the control samples.


RESULTS
The regression coefficient for the standard curves and the amplification efficiencies of the cytokine and normalizer RNA indicated that the quantification was reliable over a broad concentration range of input RNA. Relative to control cells, activation of PBMCs led to increased expression of IFN-gamma (132-fold), IL-4 (8.8-fold), IL-10 (7.2-fold), and IL-12p40 (275-fold). Basal expression of IL-12p35 was also detected.


CONCLUSIONS
This approach provides several advantages over conventional assays for cytokine measurement and can be exploited in the study of the immunopathogenesis and immunity in canine leishmaniasis.
Saldarriaga
Omar A
OA
Research Service, Department of Veterans Affairs Medical Center, South Texas Veteran Health Care System, San Antonio, Texas, USA.
Travi
Bruno L
BL
Melby
Peter C
PC
D000818:Animals; D004285:Dogs; D007371:Interferon-gamma / Q000032:analysis* / Q000096:biosynthesis / Q000235:genetics; D007378:Interleukins / Q000032:analysis* / Q000096:biosynthesis / Q000235:genetics; D007898:Leishmaniasis, Visceral / Q000276:immunology; D007963:Leukocytes, Mononuclear / Q000737:chemistry; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction
D007378:Interleukins; D012333:RNA, Messenger; D007371:Interferon-gamma