Absolute quantification of murine interleukine-4, interleukine- 10 and interferon-γ gene transcripts using Real Time PCR

Esmaelizad, M.; Jafari, S.; Rafiei, Alireza

doi:10.7508/ari.2015.02.007

Absolute quantification of murine interleukine-4, interleukine- 10 and interferon-γ gene transcripts using Real Time PCR

Authors

¹ Central Laboratory Department, Razi Vaccine and Serum Research Institute, Karaj, Iran

² Central Laboratory Department, Razi Vaccine and Serum Research Institute, Karaj, Iran Molecular Cell Biology Research Center, Mazandaran University of Medical Sciences, Sari, Iran

³ Molecular Cell Biology Research Center, Mazandaran University of Medical Sciences, Sari, Iran

10.7508/ari.2015.02.007

Abstract

The study of cytokines gene expression is quite important in various conditions of health and disease for the
evaluation of clinical responses to new vaccination approaches. An absolute quantification is based on a
calibration curve and production of standard controls to achieve more reliable results than in relative
system. In this study we attempted to construct standard controls to evaluate the murine immune response.
The number of 10 Balb/c mice immunized with hydatid cyst fluid subcutaneously with two week intervals
to induce transcription of Th1 and Th2 related cytokines. Three Pairs of primers were designed to
amplification of interleukine-4, interleukine-10 and interferon-γ by Oligo software. Partial sequences of
three cytokine genes were cloned into pTZ57T vector. Three recombinant plasmids were purified and serial
dilutions were prepared. Real time qPCR carried out using SYBR-Green I fluorescence dye and standard
curves were provided by the 7500 ABI SDS software based on the exact concentration of dilutions and the
amplification plots. Results showed that this method was able to evaluate the cytokines mRNA levels less
than 0.01pg (~150 copy). We concluded that absolute real-time qPCR can be successfully applied to the
quantification of antigen-induced cytokines.

Keywords