The use of real-time reverse transcriptase PCR for the quantification of cytokine gene expression

Abstract

Real-time reverse transcriptase polymerase chain reaction (RT-PCR) is becoming a widely used method to quantify cytokines from cells, tissues, or tissue biopsies. The method allows for the direct detection of PCR product during the exponential phase of the reaction, combining amplification and detection in a single step. Using TaqMan chemistry (Applied Biosystems, Foster City, CA) and the ABI Prism 7700 Sequence Detection System (Applied Biosystems), we validated a large panel of murine and human cytokines, as we as other factors playing a role in the immune system, such a chemokines and apoptotic markers. Although the method allows fast, sensitive, and accurate quantification, different control assays are necessary for the method to be reliable. By construction of complementary DNA (cDNA) plasmid clones, standard curves are generated that allow direct quantification of every unknown sample. Furthermore, the choice of a reliable housekeeping gene is very important. Finally, co-amplification of contaminating genomic DNA is avoided by designing sets of primers located in different exons or or intron-exon junctions. In conclusion, the real-time RT-PCF technique is very accurate and sensitive, allows high through put, and can be performed on very small samples. The development of real-time RT-PCR has resulted in an exponential increase in its use over the last couple of years, and the method has undoubtedly become the standard for quantifying cytokine patterns, clarifying many functional properties of immune cells and their associated diseases.

FIGURE 1

Schematic representation of the TaqMan principle. A: Primers and probe anneal to the target gene. Fluorescence emission does not occur because the probe is still intact. B: During the extension phase of the PCR reaction, the probe is cleaved by the 5′–3′ exonuclease activity of the Taq polymerase, allowing fluorescence emission. FW, forward; RV, reverse; F, fluorophore; Q, quencher dye.

FIGURE 2

PCR amplification plot. Fluorescence emission is measured continuously during the PCR reaction and ▵Rn (increase in fluorescence emission, from which the background fluorescence signal is subtracted) is plotted against cycle number. The threshold cycle (Ct) is the cycle at which the fluorescence exceeds a chosen threshold.

FIGURE 3

Standard curves for IL2, IL4, and β-actin (ACT). Threshold cycle values are plotted against input cDNA copy number. The differences in slope and y-intercept for the cytokine standard curves compared with the actin standard curve indicate a difference in amplification efficiency and sensitivity, respectively. If the amplification efficiencies were identical, the three standard curves should have the same slope and should be superimposed.