49, 290 –296 (1999) Copyright © 1999 by the Society of Toxicology
TOXICOLOGICAL SCIENCES
Quantitative Polymerase Chain Reaction Using an External Control mRNA for Determination of Gene Expression in a Heterogeneous Cell Population Michio Shibata,* ,1 Takeshi Hariya,* Masato Hatao,† Takao Ashikaga,‡ and Hideyuki Ichikawa* *Shiseido Skin Biology Research Laboratories, †Materials Research Laboratories, and ‡Safety Research Laboratories, Yokohama, Japan Received July 16, 1998; accepted January 7, 1999
Gene expression can be evaluated quantitatively by conventional RT-PCR or Northern blotting with the aid of a correction based on the expression of an internal control gene. However, this approach is not suitable for quantitating gene expression in a group of heterogeneous cell subsets, because the internal control gene expression may vary among the subsets. Therefore, we developed a new method for quantitative PCR using rat poly(A) 1 RNA as an external control. We used this method to investigate cytokine gene expression in lymph node cells from mice during the induction of contact hypersensitivity. Expression of the murine glyceraldehydephosphate dehydrogenase (GAPDH) gene, a candidate internal control, was not constant in cells from trinitrochlorobenzene- and vehicle-applied animals, suggesting that GAPDH gene expression changes in heterogeneous lymph node-cell subsets during induction of contact hypersensitivity. Therefore, we decided to use rat GAPDH mRNA as an external control. Cytokine gene expression was measured by quantitative PCR and was corrected based on external rat GAPDH cDNA. The reliability of this quantitative PCR was superior to that of the conventional method with an internal control. Key Words: quantitative PCR; external control; glyceraldehydephosphate dehydrogenase; contact hypersensitivity; cytokine.
A sensitive and accurate method for determination of gene expression is widely required for studies in biological sciences including toxicology. The competitive-PCR method using external-control RNA is known to be quantitative (Izutani et al., 1994), but this method is complicated and time-consuming. Recently, a quantitative PCR method, utilizing Thermus aquaticus DNA polymerase and a fluorescein-labeled probe with the ABI PRISM TM 7700 sequence detector system, was reported (Gibson et al., 1996; Holland et al., 1991). We applied this system to the determination of murine cytokine gene expression in lymph node cells (LNC) to investigate the allergenicity of chemicals. For contact allergenicity testing, a local lymph node assay (LLNA) has been proposed as an alternative 1 To whom correspondence should be addressed at Shiseido Skin Biology Research Laboratories, 2–12–1 Fukuura, Kanazawa-ku, Yokohama 236-8643, Japan. Fax: 181-45–788 –7277. E-mail:
[email protected].
to guinea pig models of contact hypersensitivity, with respect to the proliferative activity of LNC obtained from mice after topical application of test chemicals (Basketter and Scholes, 1992; Basketter et al., 1993; Kimber and Basketter, 1992;). We previously reported an ex vivo LLNA-evaluating IL-2 release from a lymph node cell culture by ELISA, showing that IL-2 release from lymph node cells in TNCB-treated mice increased (Hatao et al., 1995). In the present study we developed a quantitative PCR method, using rat GAPDH mRNA as an external control, to evaluate cytokine gene expression in LNC from mice painted in vivo with chemicals, in order to increase the sensitivity of ex vivo LLNA. In a series of experiments, the amounts of cytokine cDNA and an internal control, glyceraldehydephosphate dehydrogenase (GAPDH) cDNA in murine LNC were measured by quantitative PCR. However, the GAPDH gene could not be used as a control, since its expression appeared to change in heterogeneous lymph-node-cell subsets during the induction of contact hypersensitivity. Determination of gene expression in heterogeneous cell subsets of tissues excised from an animal is often needed in toxicological studies. Therefore, we devised an improved method that used rat GAPDH mRNA as the external control. MATERIALS AND METHODS Materials. Trinitrochlorobenzene (TNCB) was obtained from Tokyo Kasei Organic Chemicals (Tokyo, Japan). Rat poly(A) 1 RNA and Isogen LS were purchased from Nippon Gene (Tokyo, Japan). Moloney murine leukemia virus reverse transcriptase (M-MLV RT) and oligo(dT) primer were obtained from Life Technologies, Inc. (Rockville, MD). Ribonuclease inhibitor was obtained from Takara (Tokyo, Japan). PCR buffer, dATP, dUTP, dGTP, dCTP, Nuracylglycosylase, AmpliTaq TM DNA polymerase and fluorescein Taqman TM probes were obtained from PE Applied BioSystems (Foster City, CA). The sequences of primers and probes for quantitative PCR were constructed from cytokine mRNA sequences previously reported (Fort et al., 1985; Gray and Goeddel, 1983; Kashima et al., 1985; Lee et al., 1986; Moore et al., 1990; Sabath et al., 1990; Schoenhaut et al., 1992; Tso et al., 1985), and are shown in Table 1. Probes of murine cytokines and rat GAPDH were labeled with a reporter fluorescein dye, 6-carboxyfluorescein (FAM), at the 59 end and with a quencher fluorescein dye, 6-carboxytetramethylrhodamine (TAMRA), at the 39 end followed by the phosphorylation site p. Animals. Female C3H/HeN mice (Japan SLC Inc., Shizuoka, Japan), 6- to 8-weeks-old, were used throughout the experiments. Animal experiments in
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TABLE 1 Sequences of primers and probes used in the quantitative polymerase chain reaction
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our laboratory are conducted in accordance with the guidelines for animal experiments of the National Institutes of Health. The experimental protocol of this study was approved by the institutional review board for animal welfare.
obtained by quantitative PCR. By using this method, quantitative measurement of cytokine gene expression was possible over the range of 10 2 to 10 8 copies in the sample.
Test chemical application. Twenty-five microliters of TNCB solution or the vehicle, acetone-olive oil (4:1, v:v), was applied to both ears of mice once a day for 3 consecutive days. Five days after the first application, the test chemical was applied again. On the next day, the auricular lymph nodes were excised and LNC were obtained.
Stimulation index. The equation used to calculate the stimulation index (SI), i. e., the ratio of gene expression in whole LNC culture obtained from TNCB-treated mice to that in the culture from vehicle-treated mice, is shown in Figure 2. The use of this index provides correction for losses of mRNA in the sample during operations such as chloroform extraction, isopropanol precipitation, ethanol precipitation, and so on, in the total RNA preparation.
Preparation of lymph node cell suspension. Isolated lymph nodes were mechanically disaggregated and filtered through sterile gauze to obtain a single-cell suspension. This lymph-node-cell suspension was washed twice in PBS and resuspended in RPMI-1640 medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum, 25 mM HEPES, 100 mg/ml penicillin, and 100 units/ml streptomycin at a concentration of 5 3 10 6 cells/ml. An aliquot of 200 ml of lymph-node-cell suspension was seeded into each well of a 96-well tissue culture plate, and cultured for 24 or 48 h in an incubator at 37°C in an atmosphere of 5% CO 2 in air. Total RNA preparation and cDNA synthesis. After incubation, LNC were collected by pipetting from the plate, and centrifuged at 2000 3 g for 5 min. One milliliter of Isogen LS was added to the obtained LNC. Five nanograms of rat poly(A) 1 RNA was added, as an external control for determination of cytokine gene expression, to the solubilized LNC (Chomczynski and Sacchi, 1987). Then, 200 ml of chloroform was added to the extract, and the tube was shaken vigorously for 15 s. The suspension was centrifuged for 15 min at 12,000 3 g, and the RNA in the aqueous phase was transferred to another tube. The RNA was precipitated with 500 ml of isopropanol for 10 min and collected by centrifugation for 10 min at 12,000 3 g. It was taken up in 1 ml of 75% ethanol, and the ethanol suspension was centrifuged for 5 min at 12,000 3 g. The total RNA thus precipitated was dried and dissolved in ribonuclease-free sterilized water. We synthesized cDNA from 1 mg of total RNA by the use of 200 U of M-MLV RT in 20 ml of reaction mixture containing 0.5 mg of oligo(dT) primer, 50 mM Tris–HCl, pH 8.3, 75 mM KCl, 3 mM MgCl 2, 10 mM dithiothreitol, 0.25 mM dATP, 0.25 mM dTTP, 0.25 mM dGTP, 0.25 mM dCTP, and 50 U of ribonuclease inhibitor at 37°C for 1 h. Quantitative PCR. The amounts of cytokine cDNA and rat GAPDH cDNA in the test sample or vehicle control were measured by quantitative PCR using a 59 nuclease assay and an ABI PRISM TM 7700 sequence detector (PE Applied BioSystems, Foster City, CA) (Gibson et al., 1996; Holland et al., 1991). The reaction mixture was as follows: PCR buffer, 3.5 mM MgCl 2, 0.3 mM forward primer, 0.3 mM reverse primer, 0.3 mM fluorescein probe, 0.2 mM 0.2 mM dATP, 0.4 mM dUTP, 0.2 mM dGTP, 0.2 mM dCTP, 0.5 U of uracyl N-glycosylase, which prevents carry-over contamination from previously amplified DNA (Loewy et al., 1994), and 1.25 U of AmpliTaq TM DNA polymerase. The PCR conditions were: 50°C for 2 min; 95°C for 10 min; 40 cycles of 95°C for 15 s, and 60°C for 1 min. Preparation of standard template for quantitative PCR. Cytokine cDNA in LNC obtained from TNCB-treated mice or rat cDNA in rat poly(A) 1 RNA was amplified by PCR using cytokine- or rat GAPDH-specific primers. The PCR product was electrophoresed in a 2% NuSieve GTG-agarose (FMC BioProducts, Rockland, ME) and the amplicon band was excised from the gel. The gel was melted at 67°C and 3 volumes of 10 mM Tris buffer (pH 8.0) containing 1 mM EDTA (TE buffer) was added at 67°C. The dissolved amplicon was extracted with phenol and phenol-chloroform (1:1, v:v), followed by ethanol-precipitation. The precipitate was dried and dissolved in distilled water. The amount of purified amplicon was determined from the absorbance of 260 nm. An amplicon amount of 100 to 10 8 copies per reaction tube was used to obtain the calibration curve. A relative fluorescence-emission threshold was set, based on the increase of the fluorescence baseline during 3 to 10 cycles. The algorithm calculated the cycle at which each PCR amplification reached the threshold of significance, i.e., usually 10 times the standard deviation of the baseline. The calculated threshold cycle (Ct) was proportional to the number of target copies present in the sample (Gibson et al., 1996). Figure 1 shows the amplification plots and standard curves of IL-2 and IL-4
RESULTS AND DISCUSSION
We employed quantitative PCR to measure the amounts of IL-2 cDNA and murine GAPDH cDNA in murine lymph node cells during the induction of contact hypersensitivity (Fig. 3). IL-2 gene expression was markedly increased in a sensitizerdose-dependent manner (Fig. 3a). Several investigators have reported that IL-2 gene expression is increased during the induction of contact hypersensitivity induced by various sensitizers including TNCB (Cher and Mosmann, 1987; Fujisawa et al., 1996). The GAPDH gene is constitutively expressed and has been widely used as an internal control in conventional RT-PCR to correct the observed expression of a target gene. In our experiment, however, GAPDH gene expression was not constant, but was increased in samples from TNCB-treated mice, suggesting that GAPDH gene expression may change during the induction of hypersensitivity (Fig. 3b). If this is so, data correction based on GAPDH gene expression as an internal control will be invalid (Fig. 3c). In contact hypersensitivity LNC are activated, and they proliferate as a part of the immune response. Expression of GAPDH, an enzyme of the glycolytic pathway, may be induced, perhaps to different extents in different subsets of LNC. Therefore, we established a new method for quantitation, using rat GAPDH mRNA as an external control. Rat GAPDH was selected, since rat poly(A) 1 RNA containing GAPDH mRNA is commercially available and its mRNA sequence has been reported (Fort et al., 1985; Tso et al., 1985). The reproducibility of the external control method was validated. First, 5 ng of rat poly(A) 1 RNA was added to each of 2 tubes of Isogen extract obtained from the same sample of lymph-node-cell suspension. Total RNA was prepared, and cDNA was synthesized from 1 mg of total RNA. Thereafter, the amount of rat GAPDH cDNA was measured by quantitative PCR. The values obtained from the 2 tubes were almost the same (tube 1, 9.11 3 104 6 7989 copies/reaction tube [n, 3]; tube 2, 9.04 3 104 6 2880 copies/reaction tube [n, 3]). Next, the dose-dependency of rat poly(A) 1 RNA was examined. Several doses of rat poly(A) 1 RNA were added to Isogen-extracts from the lymph node cell suspension, and rat GAPDH cDNA was measured by this method (Fig. 4). Rat GAPDH cDNA was not detected in the absence of rat poly(A) 1 RNA, indicating that the combination of primers and probe used for rat GAPDH cDNA did not cross-react with murine GAPDH cDNA. The amount of rat GAPDH cDNA observed
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FIG. 1. Amplification plots and standard curves for IL-2 and IL-4 obtained by quantitative PCR. Cytokine cDNA in LNC obtained from TNCB-treated mice was amplified by PCR using cytokine-specific primers with a reaction mixture containing 0.2 mM dTTP in place of dUTP to prevent degradation by uracyl N-glycosylase during PCR. The obtained PCR product was electrophoresed in a 2% NuSieve GTG-agarose followed by purification of the product. Standard cDNA was diluted with distilled water and analyzed by quantitative PCR as described in Materials and Methods. (a) and (b): Relative fluorescence emission increase was plotted versus PCR cycle number. F, 10 8 copies of initial cDNA per reaction tube; E, 10 7; ■, 10 6; h, 10 5; Œ, 10 4; ‚, 10 3; 3, 10 2; 1, 0. (c) and (d): The calculated threshold cycle (Ct) was plotted against the initial number of target copies added in the PCR. Quantitative measurement of cytokine gene expression was possible over the range of 10 2 to 10 8 copies in the sample.
FIG. 2. Correction of cytokine mRNA expression data with external control rat GAPDH.
was proportional to the amount of added poly(A) 1 RNA. In order to confirm that primers and probes for various murine cytokines do not react with the rat cDNA, cDNA was synthesized from 5 ng of rat poly(A) 1 RNA without murine RNA, and the amounts of cDNAs amplified by the combinations of primers and probe used for various cytokines were evaluated (Fig. 5). The number of cytokine mRNA measured in the rat poly(A)1 RNA were below 100 copies/reaction tube, which was considerably small compared to those expressed in the lymph node of vehicle(acetone-olive oil)-treated mice such as 8.8 3 10 2 for IL-2, 3.0 3 10 4 for IL-10, 1.2 3 10 3 for IL-12 p35, 1.7 3 10 3 for IL-12 p40, and 1.7 3 10 3 copies/reaction tube for IFN-g. Moreover, the number of mRNAs below 100 cannot be determined correctly under our experimental conditions since the calibration curves were not defined below 100
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FIG. 3. Quantitative PCR of murine IL-2 and GAPDH mRNA. Twenty-five microliters of TNCB or vehicle was applied to both ears of mice followed by preparation of a lymph node cell suspension as described in Materials and Methods. cDNA was synthesized and gene expression of murine IL-2 (a) and GAPDH (b) were determined by quantitative PCR (lower graphs). PCR products were electrophoresed in 2% agarose gel (upper pictures). IL-2 gene expression was corrected by the use of GAPDH as an internal control (c).
copies of mRNAs. For the reasons above, we concluded that the number of synthesized cDNA from rat poly(A) 1 RNA could be regarded as negligible. Using this quantitative RT-PCR, we determined mRNA expression of IL-2 as Th 1 cytokine and IL-4 as Th 2 cytokine
FIG. 4. Dose-dependent increase of the amount of rat GAPDH cDNA measured by quantitative PCR. Rat poly(A) 1 RNA (0 to 50 ng) was added to Isogen extracts obtained from the same LNC. The total RNA fraction was prepared and cDNA was synthesized. Rat GAPDH cDNA in the samples was determined by quantitative PCR.
in the LNC, which consisted of heterogeneous cell subsets, such as Th 1 cells, Th 2 cells, B cells, antigen presenting cells, and so on. (Fig. 6). Cytokine gene expression was corrected
FIG. 5. Specificity of the PCR amplification of rat poly (A) 1 RNA. cDNA was synthesized from 5 ng of rat poly(A) 1 RNA without Isogen extract from murine LNC. Quantitative PCR was performed with various combinations of murine cytokine primers and probes.
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titative evaluation of the expression of these genes (data not shown). Thus, we conclude that quantitative PCR with an external control is a useful method for the determination of gene expression in a group of heterogeneous cell subsets, which is often needed in various studies in the field of toxicology. REFERENCES Asada, H., Linton, J., and Katz, S. I. (1997). Cytokine gene expression during the elicitation phase of contact sensitivity: regulation by endogenous IL-4. J. Invest. Dermatol. 108, 406 – 411. Basketter, D. A., and Scholes, E. W. (1992). Comparison of the local lymph node assay with the guinea-pig maximization test for the detection of a range of contact allergens. Food Chem. Toxicol. 30, 65– 69. Basketter, D. A., Selbie, E., Scholes, E. W., Lees, D., Kimber, I., and Botham, P. A. (1993). Results with OECD recommended positive control sensitizers in the maximization, Buehler and local lymph node assays. Food Chem. Toxicol. 31, 63– 67.
FIG. 6. Cytokine mRNA expression in murine LNC. Twenty-five microliters of TNCB or vehicle was applied to both ears of mice followed by preparation of lymph node cell suspension as described in the Materials and Methods. cDNA was synthesized, and gene expression of murine IL-2 (a) and IL-4 (b) was determined by quantitative PCR using rat GAPDH as an external control. Open column, 0.01% TNCB; dotted column, 0.1% TNCB; closed column, 1% TNCB. Each data point was constituted by the mean of duplicate samples.
based on external rat GAPDH cDNA. The stimulation index (SI) for IL-2 mRNA expression before cell culture was increased by TNCB application in a dose-dependent manner (Fig. 6a). Interestingly, the SI for IL-2 was decreased after culturing LNC for 24 h and 48 h. In contrast, SI for IL-4 was increased after 24- and 48-h cultures (Fig. 6b). These results suggest that IL-2 expression is upregulated at the early stage of contact hypersensitivity, followed by the upregulation of IL-4 expression at the late stage. This observation is similar to an in vivo finding (Mohler et al., 1990). IL-4 release from Th 2 cells is known to be important, especially at the effector phase of contact hypersensitivity induction by TNCB (Salerno et al., 1995; Asada et al., 1997). As the SI for IL-4 was markedly increased by higher concentrations of TNCB (1 or 0.1%), TNCB may activate Th 2 cells and provoke a humoral immune response in vivo, as well as a cellular immune response. Thus, we demonstrated that quantitative RT-PCR with an external control, rat GAPDH, is effective in a group of heterogeneous cell subsets. As a quantitative PCR using an external control has not previously been reported, cytokine profiles have been evaluated by conventional RT-PCR or Northern blotting. However, these methods are not quantitative. Our quantitative PCR with external control was shown to be sensitive and accurate. In addition to IL-2 and IL-4, we established combinations of primers and probe for IL-10, IL-12 p35, IL-12 p40 and interferon-g, all of which were confirmed to be effective for quan-
Cher, D. J., and Mosmann, T. R. (1987). Two types of murine helper T cell clone. II. Delayed-type hypersensitivity is mediated by TH1 clones. J. Immunol. 138, 3688 –3694. Chomczynski, P., and Sacchi, N. (1987). Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 156 –159. Fort, P., Marty, L., Piechaczyk, M., el Sabrouty, S., Dani, C., Jeanteur, P., and Blanchard, J. M. (1985). Various rat adult tissues express only one major mRNA species from the glyceraldehyde-3-phosphate-dehydrogenase multigenic family. Nucleic Acids Res. 13, 1431–1442. Fujisawa, H., Kondo, S., Wang, B., Shivji, G. M., and Sauder, D. N. (1996). The role of CD4 molecules in the induction phase of contact hypersensitivity cytokine profiles in the skin and lymph nodes. Immunology 89, 250 –255. Gibson, U. E. M., Heid, C. A., and Williams, P. M. (1996). A novel method for real time quantitative RT-PCR. Genome Res. 995-1001. Gray, P. W., and Goeddel, D. V. (1983). Cloning and expression of murine immune interferon cDNA. Proc. Natl. Acad. Sci. U S A 80, 5842–5846. Hatao, M., Hariya, T., Katsumura, Y., and Kato, S. (1995). A modification of the local lymph node assay for contact allergenicity screening: Measurement of interleukin-2 as an alternative to radioisotope-dependent proliferation assay. Toxicology 98, 15–22. Holland, P. M., Abramson, R. D., Watson, R., and Gelfand, D. H. (1991). Detection of specific polymerase chain reaction product by utilizing the 59339 exonuclease activity of Thermus aquaticus DNA polymerase. Proc. Natl. Acad. Sci. U S A 88, 7276 –7280. Izutani, R., Ohyanagi, H., and MacDermott, R. P. (1994). Quantitative PCR for detection of femtogram quantities of interleukin-8 mRNA expression. Microbiol. Immunol. 38, 233–237. Kashima, N., Nishi-Takaoka, C., Fujita, T., Taki, S., Yamada, G., Hamuro, J., and Taniguchi, T. (1985). Unique structure of murine interleukin-2 as deduced from cloned cDNAs. Nature 313, 402– 404. Kimber, I., and Basketter, D. A. (1992). The murine local lymph node assay: A commentary on collaborative studies and new directions. Food Chem. Toxicol. 30, 165–169. Lee, F., Yokota, T., Otsuka, T., Meyerson, P., Villaret, D., Coffman, R., Mosmann, T., Rennick, D., Roehm, N., Smith, C., Zlotnik, A., and Arai, K.-I. (1986). Isolation and characterization of a mouse interleukin cDNA clone that expresses B-cell stimulatory factor 1 activities and T-cell- and mast-cell-stimulating activities. Proc. Natl. Acad. Sci. U S A 83, 2061–2065. Loewy, Z. G., Mecca, J., and Diago, R. (1994). Enhancement of Borrelia burgdorferi PCR by uracil N-glycosylase. J. Clin. Microbiol. 32, 135–138.
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Mohler, K. M., and Butler, L. D. (1990). Differential production of IL-2 and IL-4 mRNA in vivo after primary sensitization. J. Immunol. 145, 1734 – 1739. Moore, K. W., Vieira, P., Fiorentino, D. F., Trounstine, M. L., Khan, T. A., and Mosmann, T. R. (1990). Homology of cytokine synthesis inhibitory factor (IL-10) to the Epstein-Barr virus gene BCRFI. Science 248, 1230 –1234. Sabath, D. E., Broome, H. E., and Prystowsky, M. B. (1990). Glyceraldehyde-3-phosphate dehydrogenase mRNA is a major interleukin 2induced transcript in a cloned T-helper lymphocyte. Gene 91, 185– 191.
Salerno, A., Dieli, F. G. S., Bellavia, A., and Asherson, G. L. (1995). Interleukin-4 is a critical cytokine in contact sensitivity. Immunology 84, 404 – 409. Schoenhaut, D. S., Chua, A. O., Wolitzky, A. G., Quinn, P. M., Dwyer, C. M., McComas, W., Familletti, P. C., Gately, M. K., and Gubler, U. (1992). Cloning and expression of murine IL-12. J. Immunol. 148, 3433–3440. Tso, J. Y., Sun, X. H., Kao, T. H., Reece, K. S., and Wu, R. (1985). Isolation and characterization of rat and human glyceraldehyde-3-phosphate dehydrogenase cDNAs: Genomic complexity and molecular evolution of the gene. Nucleic Acids Res. 13, 2485–2502.
