Rat pro-inflammatory cytokine and cytokine related mRNA quantification by real-time polymerase chain reaction using SYBR green

Abstract

Background: Cytokine mRNA quantification is widely used to investigate cytokine profiles, particularly in small samples. Real-time polymerase chain reaction is currently the most reliable method of quantifying low-level transcripts such as cytokine and cytokine receptor mRNAs. This accurate technique allows the quantification of a larger pattern of cytokines than quantification at the protein level, which is limited to a smaller number of proteins.

Results: Although fluorogenic probes are considered more sensitive than fluorescent dyes, we have developed SYBR Green real-time RT-PCR protocols to assay pro-inflammatory cytokines (IL1a, IL1b and IL6, TNFa), cytokine receptors (IL1-r1, IL1-r2, IL6-r, TNF-r2) and related molecules (IL1-RA, SOCS3) mRNA in rats. This method enables normalisation against several housekeeping genes (beta-actin, GAPDH, CypA, HPRT) dependent on the specific experimental treatments and tissues using either standard curve, or comparative CT quantification method. PCR efficiency and sensitivity allow the assessment of; i) basal mRNA levels in many tissues and even decreases in mRNA levels, ii) mRNA levels from very small samples.

Conclusion: Real-time RT-PCR is currently the best way to investigate cytokine networks. The investigations should be completed by the analysis of genes regulated by cytokines or involved in cytokine signalling, providing indirect information on cytokine protein expression.

Figure 1

Representative standard curves for TNFa and SOCS3 mRNAs using the LightCycler device. RNA was isolated using spleen cell cultures stimulated with LPS. The primer sets are listed in Tables 1, 2 and amplification conditions are described in "methods". Slopes and statistical value are assessed using LightCycler 3 software. Data are means ± SEM. A) Variation of standard curve for TNFa mRNA quantification according to mRNA extraction: (blue diamond) anion exchange resin RNA extraction (RNeasy mini, QIAGEN), (red diamond) phenol extraction [26]. Each value is the average of four (phenol) or three (RNeasy) independent mRNA quantifications. Only a weak difference is observed in PCR efficiencies between the templates (-3.398 and -3.399). The C_T variation is probably due to the difference in mRNA purity between both extraction methods. B) Intra-assay variation for SOCS3 mRNA quantification. Each value is the mean of three repetitions in the same experiment.

Figure 2

PCR efficiency of cytokines and related molecules among rat organs and structures. Slope values are related to PCR efficiencies (Ex) using the equation Ex = (10^-1/slope) - 1. The mean efficiency is 0.965 ± 0.085. The red line matches PCR efficiency of 1 (slope = -3.32). Slope values are assessed using LightCycler 3 software (ROCHE), from templates coming from two brain structures (hypothalamus, hippocampus) and five organs (ileum, liver, lung, spleen, skin). RNA was isolated using phenol RNA extraction. The primer sets are listed in Tables 1, 2 and amplification conditions are described in "methods". Each value is the mean of fourteen independent experiments. Data are means ± SEM.

Figure 3

Expression levels of cytokines and related molecules in control rats. A) Cycle threshold (C_T) values of pro-inflammatory cytokines and SOCS3 mRNA in various structures and organs in Lewis rats. Quantifications were carried out from 30 ng of RNA (phenol extraction). Each value is the mean of seven values. Data are means ± SEM. B) C_T values of pro-inflammatory cytokine and receptor mRNAs in hypothalamus from Sprague-Dawley rats. Quantification was carried out from 30 ng of RNA (RNeasy extraction; QIAGEN). Each value is the mean of seven values. Data are means ± SEM.

Figure 4

Validation of the 2-ΔCT method for cytokine mRNAs quantification. The ΔC_T method for relative quantification requires that the efficiency of targets (cytokines and related molecules) and reference (CypA) amplified in different tubes is approximately the same. Serial dilutions of cDNA were amplified by real-time PCR using gene-specific primers sets. The difference (ΔC_T) between cycle threshold (C_T) from target and reference was calculated for each dilution. The absolute value of the slope of ΔC_T in relation to the logarithm of cDNA concentration should be less than 0.1 [14]. Panel A shows the results of an IL1b/CypA assay where a cDNA preparation was diluted over a 10.000-fold range in three independent experiments. The data were fit using least-squares linear regression analysis (n = 3). Data are means ± SEM. Panel B: The validity of the ΔC_T method was controlled for each experiment: using a 10-fold range standard curve using sample pools as calibrators (green diamond) IL1a/CypA, (blue diamond) IL6/CypA, (red diamond) SOCS3/CypA, (purple diamond) TNFa/CypA.

Figure 5

Quantification of cytokine-related molecule mRNAs in various pathophysiological conditions. Studies were in accordance with French ethical guidelines and approved by a Medical Committee of the French Army Medical Research Centre (CRSSA). Sham-animals were handled in the same conditions as injured animals without injury exposure. Conditions for 40% total body surface thermal injury (TI) and 8 Gy whole-body irradiation (WBI) were described previously [12,22]. A 30 minutes contention was achieved in the irradiation device. Data are means ± SEM, n = 6. Statistical significance was assessed using ANOVA or Student's t-test (* p < 0.05; ** p < 0.01; *** p < 0.001). A.) 1; increase in spleen TNFa mRNA 3 h after 8 Gy WBI, 2; increase in liver SOCS3 mRNA 24 h after 40% total body surface TI, 3; ileum increase in IL1RA mRNA 23 h after 8 Gy WBI. B.) 1; increase in hypothalamus IL1a mRNA 3 h after a 30 minutes contention, 2; decrease in liver IL6 mRNA 6 h after 40% total body surface TI, 3; increase in hypothalamus IL6-receptor mRNA 2 h after 8 Gy WBI.