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Technical Briefs |
1
University Department of Laboratory Medicine, University of Milano-Bicocca, Hospital of Desio, Via Mazzini 1, 20033 Desio, Milan, Italy;
2
Department of Laboratory Medicine, Hospital of Merate, Largo Mandic 1, 23807 Lecco, Italy;
aauthor for correspondence: fax 39-036-2383464, e-mail mocarelli{at}uds.unimb.it)
The aromatic hydrocarbon (Ah) gene battery (1) comprises Ah receptor (AHR), its nuclear translocator (ARNT), and the genes that encode the detoxifying enzymes of phase I (e.g., cytochrome P4501A1, -1A2, and -1B1) and phase II. These enzymes and growth regulatory factors such as interleukin 1-ß (IL-1ß) are coordinately induced by polychlorinated dibenzo-p-dioxins and polycyclic aromatic hydrocarbons, toxic chemicals that are ubiquitous in ambient air and in cigarette smoke, via AHR (1)(2)(3)(4). Quantification of the regulation of AHR and related genes could be useful to monitor the degree of polycyclic aromatic hydrocarbon exposure and to understand the pathways to chemical carcinogenesis and atherosclerosis (5). The expression of AHR and related genes has been studied using Northern blot analysis (6) or quantitative reverse transcription-PCR (RT-PCR) using labeled primers (7), which are low-sensitivity techniques; competitive RT-PCR (8), which is more sensitive but quite laborious; and real-time PCR (9), which is expensive.
An interesting technique, proposed for the first time by Tsai and Wiltbank (10) and considered later by Bor et al. (11), is called "calibrated " RT-PCR; it is a competitive RT-PCR based on the addition of a fixed amount of internal standard (IS) to the sample and to a set of calibrators. The labor, time, and costs for this method are lower than those for classic competitive RT-PCR. We present calibrated RT-PCR assays to study the expression of AHR, ARNT, cytochrome P4501B1 (CYP1B1), and IL-1ß for the AHR gene battery and of ß-actin (ß-ACT) as housekeeping gene in peripheral blood mononuclear cells from 32 healthy subjects.
Total RNA was extracted according to the method of Chomczynski and Sacchi (12) from 1 x 107 peripheral blood mononuclear cells in suspension isolated by step-gradient centrifugation on Histopaque. RNA was resuspended in 50 µL of sterile water, quantified by measuring the absorbance at 260 nm, diluted to 100 mg/L with sterile water, and stored in liquid nitrogen.
The ISs, designed to be smaller than the corresponding target sequences, were homologous recombinant cDNAs obtained by oligonucleotide-mediated mutagenesis (13) from AHR, ARNT, CYP1B1, and ß-ACT sequences inserted in pGEM plasmid. A more recently developed and simple method (14)(15) was adopted to obtain the IL-1ß IS. Table 1
shows the primers used for the amplification and the oligonucleotides used to produce corresponding ISs. Forward and reverse primers were chosen on different exons to include the intervening sequences when the DNA was amplified. Plasmids containing ISs were linearized and transcribed into RNA using the RiboMAXTM Large Scale Production System T7 (Promega) followed by an RNase-free DNase treatment to remove the DNA template. ISs were purified according to the technical manual of RiboMAXTM. RNA was diluted to the desired concentrations in sterile water. Ribosomal RNA 16S–23S from Escherichia coli was added (final rRNA concentration, 100 mg/L) and stored in liquid nitrogen. The rRNA from E. coli did not interfere with RT-PCR.
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Competitive RT-PCR was performed as described previously (16). Gene-specific PCR reagents and conditions were as follows: (a) MgCl2, 1.5 mM for ARNT and CYP1B1 and 2 mM for AHR, IL-1ß, and ß-ACT; (b) Taq polymerase, 1.25 U for AHR, ARNT, and CYP1B1 and 2.5 U for IL-1ß and ß-ACT; (c) annealing-extension temperature, 59 °C for IL-1ß, 60 °C for ß-ACT and AHR, 63 °C for ARNT and CYP1B1; (d) cycles numbers, 33 for AHR, 35 for ARNT, CYP1B1, and ß-ACT, 37 for IL-1ß. Quantification was performed as described previously (16)(17), with a correction factor of 1.28 for AHR, 1.2 for ARNT and IL-1ß, 1.19 for CYP1B1, and 1.24 for ß-ACT, accounting for the difference in size of the target:IS ratio.
Calibrated RT-PCR was performed according to Bor et al. (11) by adding a fixed amount of IS to the unknown samples and to a set of calibrators containing 0.35, 0.67, 6.03, 0.097, and 2939 amol of mRNA in 260 ng of total RNA, respectively, for AHR, ARNT, CYP1B1, IL-1ß, and ß-ACT. Serial dilutions were prepared from each first calibrator to obtain calibrators with lower concentrations (220, 180, 140, 100, 50, 10, and 5 ng of total RNA). A calibration curve was constructed by plotting the ratio, calibrator mRNA:RNA IS, against the amount of calibrator mRNA. The amplified target and the IS were separated by electrophoresis. The mRNA concentrations in the unknown samples were calculated from the calibration curve based on the ratio between the sample and the corresponding RNA IS. The reverse transcription and PCR conditions used were the same as those for the competitive RT-PCR.
Quantification of calibrated RT-PCR was performed as described previously (11), using the above-mentioned correction factors.
The target:IS ratio results were independent of the number of cycles (25–40) of amplification, indicating that, for all genes, the relative amounts of the two amplified products stayed identical throughout the PCR amplification exponential phase and when the plateau phase was reached.
An example of an agarose gel showing the calibration curve for calibrated RT-PCR of AHR performed with a constant amount of RNA IS (0.166 amol) is shown in Fig. 1A
. The calibration curve is linearly described by the equation y = ax + b, where x is the calibrator total RNA and y represents the ratio of the target product to the IS product (Fig. 1B
). This equation was determined for each assay and used to detect the amount of sample mRNA. One sample for AHR and 10 samples for IL-1ß exceeded the calibration curve and were tested using calibration curves covering a higher linear range (up to 500 ng of total RNA for AHR and up to 1000 ng for IL-1ß). We preferred to use a higher concentration of IS instead of diluting the sample to maintain the characteristics of the sample.
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The calibrated RT-PCR showed good intraassay imprecision; the CV (calculated from 10 replicates of a sample) was 7.3%, 6.8%, 8.7%, 6.0%, and 6.9% for AHR, ARNT, CYP1B1, IL-1ß, and ß-ACT, respectively. Interassay imprecision was calculated from results of duplicate RT-PCRs in two different analytical runs on the same RNA preparation of 32 subjects; the CV was 10%, 8.5%, 11%, 8.0%, and 8.0% for AHR, ARNT, CYP1B1, IL-1ß, and ß-ACT, respectively. These results are similar to values reported previously (11).
The calibrated RT-PCR is suitable for a wide range of measurements; it therefore can be used to quantify genes whose expression can vary widely in different subjects. The expression of AHR and related genes, expressed as amol mRNA/100 ng of total RNA, was 0.012–0.55 for AHR, 0.07–0.36 for ARNT, 0.02–6.14 for CYP1B1, 0.015–0.33 for IL-1ß, and 378-1940 for ß-ACT. A higher sensitivity can be obtained by increasing the number of PCR cycles, as we did with the IL-1ß gene, whose expression was lower respect to the other investigated genes, but keeping in mind that heteroduplex formation can occur with an excessive number of cycles.
Samples from 30 different subjects were analyzed with both competitive RT-PCR and calibrated RT-PCR assays for all genes tested. Weighted Deming regression analysis was performed on all data, and the regression coefficient (R), slope (95% confidence interval), and y-intercept (95% confidence interval) for each gene was as follows: for AHR, R = 0.951, slope = 0.90 (0.7–1.05), y-intercept = 0.05 amol (-1.22 to 1.32 amol); for ARNT, R = 0.912, slope = 1.0 (0.7–1.3), y-intercept = -2.09 amol (-6.1 to 1.9 amol); for CYP1B1, R = 0.948, slope = 1.14 (0.9–1.4), y-intercept = -0.22 amol (-2.19 to 1.73 amol); for IL-1ß, R = 0.967, slope = 1.05 (0.9–1.2), y-intercept = 0.1 amol (-0.53 to 0.73 amol); for ß-ACT, R = 0.961, slope = 1.1 (0.9–1.3), y-intercept = -0.37 amol (-1.0 to 0.28 amol).
The results obtained with calibrated RT-PCR were in agreement with those obtained with classic competitive RT-PCR for all tested genes, showing a good correlation between the two techniques.
Compared with the competitive RT-PCR, calibrated RT-PCR has the advantage of requiring a smaller amount of biological sample (one replicated tube instead of six single tubes), which is important when the amount of the sample is a limiting factor, and allows reduced labor, time, and costs. The amount of mRNA for the target genes is strictly dependent on the corresponding RNA IS preparation, a procedure that has an intrinsic variability associated with in vitro transcription, purification from nucleotide, and quantification. Different from the method of Bor et al. (11), where the IS and target RNA share only the sequences of the primer sites, our synthetic RNA has a sequence identical to that of the target mRNA except the deleted region so that there are no differences in amplification efficiencies; the difference in size between mimic and target allows separation of the corresponding amplification products in an agarose gel. We preferred to clone the IS fragments into a vector to facilitate their spectrophotometric quantification, as reported by Tsai and Wiltbank (10). False-positive results attributable to the amplification of DNA potentially present in samples after the RNA extraction process were avoided by an appropriate choice of primer sequences in two different exons.
To minimize variability, it is advisable to prepare a sufficient quantity of RNA IS that can be used for analyses over a longer period. Our RNA IS, added to a larger amount (100 mg/L) of ribosomal RNA to prevent RNA degradation and stored in liquid nitrogen, showed good stability over time. Quantification with this technique can potentially be applied to study gene expression in every tissue that allows RNA extraction.
In conclusion, calibrated RT-PCR is a reliable and accurate method for evaluating the expression of AHR and a related battery of genes that have been proposed as mediators of the majority of biochemical and toxicologic effects of several xenobiotics.
Acknowledgments
This work was supported by Grant 2896 from Regione Lombardia (Milan, Italy). We thank Patrizia Pighi, Raffaella Fioratto, and Paola D’Alessandro for helpful contributions.
References
in human uterine endometrial adenocarcinoma RL95-2 cells. Biochem Biophys Res Commun 1997;238:338-342.[ISI][Medline]
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