CyProQuant-PCR: a real time RT-PCR technique for profiling human cytokines, based on external RNA standards, readily automatable for clinical use

Abstract

Background: Real-time PCR is becoming a common tool for detecting and quantifying expression profiling of selected genes. Cytokines mRNA quantification is widely used in immunological research to dissect the early steps of immune responses or pathophysiological pathways. It is also growing to be of clinical relevancy to immuno-monitoring and evaluation of the disease status of patients. The techniques currently used for "absolute quantification" of cytokine mRNA are based on a DNA standard curve and do not take into account the critical impact of RT efficiency.

Results: To overcome this pitfall, we designed a strategy using external RNA as standard in the RT-PCR. Use of synthetic RNA standards, by comparison with the corresponding DNA standard, showed significant variations in the yield of retro-transcription depending the target amplified and the experiment. We then developed primers to be used under one single experimental condition for the specific amplification of human IL-1beta, IL-4, IL-10, IL-12p40, IL-13, IL-15, IL-18, IFN-gamma, MIF, TGF-beta1 and TNF-alpha mRNA. We showed that the beta-2 microglobulin (beta2-MG) gene was suitable for data normalisation since the level of beta2-MG transcripts in naive PBMC varied less than 5 times between individuals and was not affected by LPS or PHA stimulation. The technique, we named CyProQuant-PCR (Cytokine Profiling Quantitative PCR) was validated using a kinetic measurement of cytokine transcripts under in vitro stimulation of human PBMC by lipopolysaccharide (LPS) or Staphylococcus aureus strain Cowan (SAC). Results obtained show that CyProQuant-PCR is powerful enough to precociously detect slight cytokine induction. Finally, having demonstrated the reproducibility of the method, it was applied to malaria patients and asymptomatic controls for the quantification of TGF-beta1 transcripts and showed an increased capacity of cells from malaria patients to accumulate TGF-beta1 mRNA in response to LPS.

Conclusion: The real-time RT-PCR technique based on a RNA standard curve, CyProQuant-PCR, outlined here, allows for a genuine absolute quantification and a simultaneous analysis of a large panel of human cytokine mRNA. It represents a potent and attractive tool for immunomonitoring, lending itself readily to automation and with a high throughput. This opens the possibility of an easy and reliable cytokine profiling for clinical applications.

Figure 1

Primer validation. A. Agarose gel electrophoresis of several CyProQuant-PCR products generated by amplification of a pool of cDNA from PBMC stimulated in vitro by LPS or PHA. Scale is shown in base pairs (bp). B. Dissociation curve analysis of these CyProQuant-PCR products. Negative derivative of the fluorescence is plotted against temperature. The single peak shows that SYBR Green fluorescence detects only the specific CyProQuant-PCR product. C. Amplification plots and standard curve resulting from the amplification of a range of β2-MG external RNA standard using CyProQuant-PCR.

Figure 2

Comparison to TaqMan^® technology. Total RNA was extracted from isolated monocytes stimulated for 6 hours by LPS, serial-diluted 1:10 and retro-transcribed to generate standard curves by plotting the C_T against the concentration of this cellular RNA in arbitrary units. TNF-α transcripts were amplified by real time PCR using either (A) the TaqMan^® commercial kit and the TaqMan^® universal PCR master mix or (B) our primers and the SYBR Green PCR master mix. Data represent the standard curves obtained for the two techniques, their slopes and the deducted efficiencies.

Figure 3

TNF and MIF transcription and secretion kinetics. PBMC from 2 donors were stimulated for 3, 9 or 18 hours (T3, T9 and T18 respectively) with LPS or SAC. TNF-α and MIF transcripts were quantified using CyProQuant-PCR and the corresponding secreted proteins were measured by ELISA. Transcript numbers are normalised for one million copies of β2-MG RNA relative to the level of transcripts in un-stimulated cells (fold increase). Secreted proteins are expressed as pg/mL for one million living cells.

Figure 4

Early cytokine production kinetics by PBMC stimulated in vitro with LPS or SAC. Total cellular RNA was extracted from PBMC from 2 donors stimulated for 0.5, 1, 2, 3, 6 or 9 hours with LPS (blue bars) or SAC (red bars). Cytokine transcripts were quantified using CyProQuant-PCR. The results are shown for one donor and were expressed in mRNA copy numbers calculated relative to un-stimulated cells, after normalisation against β2-MG (fold increase).

Figure 5

TGF-β1 transcripts quantification in malaria patients and asymptomatic controls TGF-β1 transcripts were quantified without stimulation and after 22 hours of LPS stimulation of PBMC harvested from asymptomatic controls (n = 5) and from patients suffering from acute malaria (n = 20). Results are normalised for one million copies of β2-microglobulin. Whiskers indicate data range, boxes extend from the 25^th to the 75^th percentile and the horizontal lines show the median. Asterisk indicates significant higher value in malaria patients compared to asymptomatic controls (p = 0.036) in the capacity of response to LPS.