Calibrated user-friendly reverse transcriptase-PCR assay: quantitation of epidermal growth factor receptor mRNA

Department of Clinical Biochemistry, KH, Aarhus University Hospital, DK-8000 Aarhus C, Denmark.
^a Address correspondence to this author at: Department of Clinical Biochemistry, KH, University Hospital of Aarhus, Nørrebrogade 44, DK-8000 Aarhus C, Denmark. Fax (45) 89493060; e-mail Domain31.ak14s. akklkvb1{at}aaa.dk.

	Abstract

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We report a competitive reverse transcriptase-PCR (RT-PCR) assay and a calibrated user-friendly RT-PCR assay (CURT-PCR) for epidermal growth factor receptor (EGFR) mRNA. A calibrator was prepared from isolated rat liver RNA, and the amount of EGFR mRNA was determined by competitive RT-PCR. In CURT-PCR, a calibration curve was developed by plotting the ratio between the amount of PCR product originating from the calibrator and the RNA internal standard vs the amount of EGFR mRNA present in the calibrator. A fixed amount of RNA internal standard was coamplified with the EGFR mRNA present in the calibrator or in the sample, using the same primer set. The primers were chosen in regions of the EGFR mRNA that show 100% homology between human, rat, and mouse. The amount of EGFR in the unknown samples was calculated from the calibration curve based on the ratio between PCR product originating from the sample and the corresponding RNA internal standard. Competitive RT-PCR and CURT-PCR were used for rat liver samples from 21 different animals. Comparable results were obtained by the two methods. The imprecision of the CURT-PCR method was 8% (n = 20), and the imprecision of the traditional competitive RT-PCR was 16% (n = 17). We conclude that the CURT-PCR method developed is suitable for routine applications such as quantitation of EGFR expression in tumor biopsies. The imprecision is relatively low. Furthermore, the use of a calibration curve makes it possible to analyze a large number of samples in one analytical run and to accept or reject the results according to existing rules for quality assurance.

	Introduction

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Determination of mRNA concentrations of specific genes is becoming increasingly important as a measure of gene expression. With the recent development of reverse transcriptase-PCR (RT-PCR),¹ the sensitivity of mRNA determination has been increased dramatically. One approach to quantitate mRNA is the use of RNA internal standards that are reverse-transcribed and coamplified with the same primer set as the target sequence in the same reactions. This technique, called competitive RT-PCR, requires several reaction tubes for each sample to determine the concentration of the specific mRNA in each unknown sample (1)(2)(3)(4)(5)(6). Furthermore, it is difficult to monitor the quality of this type of analysis on a daily base. Despite these limitations, competitive RT-PCR mRNA analysis is a useful tool for research purposes, but the methodology is less suitable for the routine analysis of mRNA.

Interest in routine quantification of mRNA is increasing, for example, in connection with studying gene expression in malignant diseases. One gene of potential interest is the EGFR gene, which codes for epidermal-growth-factor receptor (EGFR), a 170-kDa protein with an intracellular tyrosine kinase domain, which plays important roles in the growth and differentiation of healthy, regenerative, and neoplastic tissues (7)(8). Overexpression of EGFR has been documented in a wide variety of human tumors, and in a number of tumors, overexpression of EGFR correlates with reduced relapse-free intervals and overall survival (9). The important role of EGFR in human malignancies has led to intensive studies of the expression of EGFR in clinics. The transcriptional and posttranscriptional regulation of EGFR expression has been studied by different methods, including radioligand binding assays (10), immunological assays (11), northern blot analysis (12), RNase protection assays (13), and competitive RT-PCR using a homologous RNA internal standard (Thøgersen et al. 1997, submitted). Furthermore, amplification of the gene coding for the c-erbB-2 receptor, which is closely related to EGFR, has been studied with a recently developed method using fluorogenic probes (TaqMan, Perkin–Elmer) (14).

In the present paper, we describe two types of EGFR mRNA analysis, a competitive RT-PCR and a calibrated user-friendly RT-PCR (CURT-PCR). The CURT-PCR is based on addition of a fixed amount of internal standard to the samples and to a set of calibrators. This design makes the analysis suitable for routine use, because the results obtained for individual samples can be accepted or rejected according to existing rules for quality assurance.

	Material and Methods

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tissue preparation and isolation of rna
The study was conducted on 21 male Wistar rats Molle-gärd Avslab), 8 weeks of age. The animals were anesthetized with intraperitoneal injections of 50 mg/kg pentobarbital before removal of the liver. The liver was removed, snap-frozen in liquid nitrogen, and stored at -80 °C. Total cellular RNA was isolated from ~40 mg homogenized rat liver with the Purescript kit (Gentra) according to the instructions provided by the manufacturer. After purification, the concentration and purity of the RNA preparation were analyzed by absorbance at 260 and 280 nm (Shimadzu, model UV-160A) (15).

preparation of rna internal standard for quantitative pcr
Construction of the internal standard was accomplished by synthesizing two primers, P1 and P2, containing sequences for the T7 promoter, the target gene (EGFR) mRNA, a spacer DNA that is a 0.6-kb EcoRI-BamHI fragment of the v-erb B oncogene (MIMIC, commercially available from Clontech) and a poly(dT) tail. Fig. 1 gives a schematic description of the construction of the standard. The primer P1 contains the T7 RNA polymerase promoter sequence, the EGFR forward primer sequence, and a region hybridizing to the spacer DNA. The other primer (P2) consists of sequences for a poly(dT)₂₀ tract, the reverse EGFR primer, and a region hybridizing to the other end of the spacer DNA. A 50-µL PCR containing 5 mmol/L MgCl₂, 2 mmol/L deoxyribonucleoside triphosphates, 1 ng of the spacer DNA, 10 pmol of each of the P1 and P2 primers, and 2.5 units of Taq DNA polymerase (Pharmacia) was performed using a Perkin–Elmer Thermal Cycler with the following cycle parameters: denature; 94 °C for 1 min; anneal 65 °C for 30 s; and extend 72 °C for 90 s. Before the PCR cycles, the reaction was initially heated to 94 °C for 3 min. After 30 cycles of amplification, the PCR products were extended at 72 °C for 7 min. Amplification was monitored by gel electrophoresis, and the presence of a single DNA band of the correct size was demonstrated. To produce the RNA internal standard, 1 µg of the generated molecule was incubated with 1 unit of T7 RNA polymerase (Boehringer Mannheim) at 37 °C for 2 h in the buffer supplied by the manufacturer. The RNA internal standard was treated with RNase-free DNase I (Promega) to remove the DNA template and extracted with water-saturated phenol/chloroform (24:1, by volume). The RNA contained in the water phase was transferred to a fresh tube and precipitated by addition of 0.1 volume of 4 mol/L NaCl and 2.5 volumes of 960 mL/L ethanol. The RNA internal standard was precipitated by centrifugation for 15 min in an Eppendorf centrifuge (14 000 rpm) and washed with 700 mL/L ethanol. The pellet was resuspended in nuclease-free water and quantitated by the absorbance at 260 nm (16)(17). The absence of DNA was demonstrated by the inability to obtain PCR amplification products in the absence of reverse transcription (data not shown).

View larger version (17K): [in this window] [in a new window]   Figure 1. Schematic presentation of the generation of the RNA internal standard. The spacer DNA fragment used was a 0.6-kb EcoRI-BamHI fragment of the v-erb B oncogene. The primers P1 and P2 were used to generate a DNA fragment that can be transcribed in vitro to yield EGFR RNA internal standard. Spacer DNA was amplified with primers containing 5' attached sequences. One primer (P1) contained the T7 RNA polymerase promoter sequence and the EGFR gene-specific primer sequence. The other primer (P2) contained the other EGFR gene-specific primer sequence and a poly(dT)20 stretch. The T7 promoter sequence allowed in vitro transcription of the PCR products, and the poly(dT) sequence on the other primer yielded an RNA standard with a poly(dA) tail. Detailed experimental procedures for generating the RNA internal standard are described in Materials and Methods. The nucleotide sequence of P1 is 5'-CCAAGCTTCTAATACGACTCACTATAGGGAGAGAGAGGAGAACTGCCAGAAGCAGATGAGTATCTTGTCCC-3'; the sequence of primer P2 is 5'-TTTTTTTTTTTTTTTTTTTTGTAGCATTTATGGAGAGTGTTGAGTCCATGGGGAGCTTT-3' (The T7 promoter sequence and the sequence composed of 20 T nucleotides are shown in italics. Underlined nucleotides represent forward and reverse gene-specific EGFR primer sequences. The regions of the primers hybridizing to the EcoRI-BamHI fragment of the v-erb B oncogene are bold. The forward and reverse gene-specific EGFR primers had the sequence 5'-GAGAGGAGAACTGCCAGAA-3' and 5'-GTAGCATTTATGGAGAGTG-3', respectively. T7, T7 promoter sequence; EGFR FP, EGFR forward primer sequence; EGFR RP, EGFR reverse primer sequence; dT, d(T)20 tail; and dA, d(A)20 tail.

quantitative rt-pcr assay
Total RNA (0.8 µg) in combination with serial dilutions of the RNA internal standard (1.86 x 10^-17, 4.65 x 10^-18, 1.16 x 10^-18, and 2.9 x 10^-19 mol, respectively) was added to a reverse transcription reaction mixture containing 50 mmol/L KCL, 10 mmol/L Tris-HCL (pH 8.3), 6.25 mmol/L MgCl₂, 1 unit/L RNase inhibitor, 1 mmol/L deoxyribonucleoside triphosphates (dATP, dCTP, dTTP, or dGTP), 2.5 µmol/L of a 16-mer d(T)₁₆ oligonucleotide primer, and 2.5 U/L reverse transcriptase (all reaction components were from Perkin–Elmer) in a total volume of 20 µL. This mixture was incubated at 42 °C for 30 min in a Perkin–Elmer 9600 thermocycler. The reverse transcription products (2.5 µL) were mixed with 22.5 µL of PCR buffer containing 50 mmol/L KCL, 10 mmol/L Tris-HCL (pH 9.0), 1.5 mmol/L MgCl_2, 0.2 mmol/L deoxyribonucleoside triphosphates (dATP, dCTP, dTTP, or dGTP), 1.25 units of Taq DNA polymerase (Pharmacia), and 25 pmol of each of the EGFR primers. The sequence of the EGFR forward primer was 5'-GAGAGGAGAACTGCCAGAA, and the sequence of the reverse primer was 5'-GTAGCATTTATGGAGAGTG. The primers (residue 761–779 and 1196–1214, respectively, of the EGFR gene) were chosen in a region with complete homology between human, rat, and mouse EGFR. All reactions were initially denatured at 94 °C for 3 min before PCR amplification. The standard cycling program was as follow: 94 °C for 1 min; 57 °C for 30 s; and 72 °C for 90 s. After 30 cycles, the PCR products were extended at 72 °C for 7 min. After completion of the PCR, 10-µL aliquots of the reaction mixtures were analyzed on a 2% ethidium-stained agarose gel. The intensity of the bands was determined by computer scanning (Gel doc 1000, Bio-Rad). The intensities of the bands were corrected for the difference in size between the PCR products for the RNA internal standard (314 bp) and EGFR mRNA (454 bp).

CURT-PCR was performed as described above except that a fixed amount of RNA internal standard (4.65 x 10^-18 mol) was added to six calibrators and the unknown samples before RT-PCR. The calibrators were prepared from a pool of liver RNA (2.7 g/L). The stock solution was diluted in water to allow the preparation of five additional calibrators (containing 2.0, 1.0, 0.5, 0.25, and 0.125 g/L total RNA). The amount of EGFR mRNA present in the stock solution of the calibrator was determined by competitive RT-PCR. A calibration curve was developed by plotting the ratio between the amount of PCR product originating from the calibrators and the RNA internal standard against the amount of EGFR mRNA present in the calibrator. The amount of EGFR mRNA in the unknown samples was calculated from the calibration curve based on the ratio between the sample and the corresponding RNA internal standard.

analysis of samples
RNA extracted from the 21 rat livers was analyzed for the content of EGFR mRNA, using both competitive RT-PCR assay and CURT-PCR. In addition, sets of samples were analyzed at two different occasions with both methods to evaluate the imprecision of the methods.

statistical methods
The degree of consistency between values obtained by CURT-PCR and competitive RT-PCR assays was determined according to the method of Bland-Altman (18) and linear-regression analysis.

	Results

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amplification efficiency of egfr mRNA AND THE INTERNAL RNA STANDARD
One requirement for the accurate quantitation of mRNA in competitive RT-PCR is that the amplification efficiency of the target and the internal standard is identical. To demonstrate that this was the case, five different samples (each of them containing 0.8 µg of total RNA) and three calibrators (containing 2.2, 1.6, or 0.8 µg of total RNA) were reverse-transcribed with a constant amount of RNA internal standard (4.65 x 10^-18 mol) to generate cDNA products. The cDNA products were then coamplified over a range of cycles. The ratio of EGFR mRNA product to RNA internal standard product remained constant with all of the cycles investigated (26, 28, 30, 32, and 34 cycles; Fig. 2 ). On the basis of those results, 30-cycle PCR was chosen for additional studies.

View larger version (17K): [in this window] [in a new window]   Figure 2. Amplification efficiency of target and internal standard RNA at different PCR cycles. Five different samples, each containing 0.8 µg of total RNA (solid symbols and solid lines), and three calibrators, each containing 2.2, 1.6, or 0.8 µg of total RNA (open symbols and dotted lines), were reverse-transcribed with a constant amount of RNA internal standard (4.65 x 10-18 mol). The cDNA products were coamplified for 26, 28, 30, 32, or 34 cycles. The PCR products were separated in a 2% ethidium bromide-stained agarose gel. The intensity of the bands was determined by computer scanning (Gel doc 1000, Bio-Rad), and the ratio of the PCR products was plotted as a function of the cycle numbers.

competitive rt-pcr
We have developed a competitive RT-PCR method that coamplifies a 454-bp sequence of EGFR and a 314-bp sequence of an RNA internal standard with the same primer set. A fixed amount of liver RNA was RT-PCR-amplified together with different concentrations of RNA internal standard. The ratio of PCR products was graphed as a function of added RNA internal standard after separation of the target and internal standard by electrophoresis (Fig. 3 ). The concentration of EGFR mRNA was then determined as the concentration of RNA internal standard that would have given the same intensity as the EGFR mRNA analyzed.

View larger version (24K): [in this window] [in a new window]   Figure 3. Competitive RT-PCR. (A) A fixed amount of liver RNA (0.8 µg) was RT-PCR-amplified together with different concentrations of RNA internal standard (1.86 x 10-17, 4.65 x 10-18, 1.16 x 10-18, or 2.9 x 10-19 mol of RNA standard, respectively). After the PCR amplification (30 cycles), 10 µL of reaction mixture was subjected to electrophoresis in a 2% agarose gel containing ethidium bromide, and the gel was photographed under ultraviolet illumination. The lower band is PCR-amplified from the standard RNA (314 bp), whereas the upper band originates from EGFR mRNA (454 bp). Lane 1, marker DNA composed of X 174 DNA digested with HaeIII; the five upper bands have the size of 1353, 1078, 872, 603, or 310 bp; lane 2, mock reaction without RNA; lane 3, reaction with pure RNA standard (1.86 x 10-17 mol); lane 4, reaction with purified RNA (0.8 µg) without standard; lanes 5–8, reaction with 1.86 x 10-17, 4.65 x 10-18, 1.16 x 10-18, or 2.9 x 10-19 mol of RNA internal standard, respectively, amplified together with 0.8 µg of sample RNA. (B) The log of the ratio of amplified target to internal standard product is graphed as a function of the log of a known amount of internal standard added to the PCR reaction. When the molar ratio of the sample and the internal standard is equal to 1, the log of the ratio is equal to 0, and the concentration in the sample can be read from the x-axis.

curt-pcr
A fixed amount of internal standard was added to a set of calibrators and the samples. A calibration curve was developed by plotting the ratio of the band intensities for the individual calibrator and the corresponding internal standard against the amount of EGFR mRNA present in the calibrator. A linear relationship covering the range from 0.19 x 10^-18 to 4.2 x 10^-18 mol EGFR mRNA/µg RNA was obtained (Fig. 4 ). The calibration curve could be described by the equation y = ax b. This equation was determined each time the assay was run and was used to determine the concentration of EGFR mRNA in the liver samples. Samples exceeding the calibration curve were diluted in water before repeated analysis.

View larger version (21K): [in this window] [in a new window]   Figure 4. Calibration curve for the CURT-PCR assay. (A) Six concentrations of calibrator (prepared from a pool of liver RNA), each containing 2.2, 1.6, 0.8, 0.4, 0.2, or 0.1 µg of calibrator RNA (which corresponds to 4.20, 3.05, 1.53, 0.76, 0.38, or 0.19 x 10-18 mol of EGFR mRNA, respectively), were coamplified with a constant amount of RNA internal standard (4.65 x 10-18 mol of RNA) for 30 cycles after reverse transcription. The PCR products were separated on a 2% agarose gel, and the intensity of the bands was determined by computer scanning (Gel doc 1000, Bio-Rad). The lower band is PCR-amplified from the standard RNA (314 bp), and the upper band originates from EGFR mRNA (454 bp). (B) Ratio of the target product to the internal standard product was plotted against the amount of calibrator RNA added to the RT-PCR

comparison of curt-pcr and competitive rt-pcr
The imprecision was determined from the analysis of samples ranging from 0.79 x 10^-18 to 8.4 x 10^-18 mol EGFR mRNA/µg RNA two times in different analytical runs. The results are shown in Fig. 5 . On the basis of the presented data, the imprecision was calculated to be 16% (n = 17) for the competitive RT-PCR assay and 8% (n = 20) for CURT-PCR. Twenty-one samples from different rat livers were analyzed with both CURT-PCR and competitive RT-PCR assays. The results obtained showed a regression line of y = 0.82x 0.53 (r = 0.91), where x represents the competitive RT-PCR method and y represents the CURT-PCR (Fig. 6 A). We assessed the degree of agreement between the two methods by plotting the difference of the results obtained vs the mean of the two methods. (Fig. 6B ). The mean difference of the two methods was -0.05, with a standard deviation of 0.40. The 95% confidence intervals for the mean difference were -0.24 and 0.13. This confidence interval included 0, indicating that there is no evidence of systematic bias. The limits of agreement were 0.75 to -0.85, which means that the difference between the two methods was between 0.8 and -0.9 for 95% of the samples.

View larger version (12K): [in this window] [in a new window]   Figure 5. Imprecision of CURT-PCR and competitive RT-PCR. (A) EGFR mRNA in rat livers (n = 17) was determined in two analytical runs by competitive RT-PCR. Differences between the two results obtained for each sample (y-axis) are plotted against the average of the two results (x-axis). The mean of the differences ± SD of bias is 0.15 ± 0.85, CV = 16%, n = 17. (B) EGFR mRNA in rat livers (n = 20) was determined in two analytical runs by CURT-PCR. The difference of the two CURT-PCR assays (y-axis) are plotted against the average of the two results (x-axis). The mean of the differences ± SD of bias is -0.01 ± 0.32, CV = 8%, n = 20.

View larger version (13K): [in this window] [in a new window]   Figure 6. Comparison of CURT-PCR and competitive RT-PCR. (A) Linear regression analysis of the competitive RT-PCR (x-axis) and the CURT-PCR (y-axis). Slope = 0.82, intercept = 0.53, r = 0.91, n = 21. (B) The differences between results obtained with the competitive RT-PCR assay and CURT-PCR (y-axis) are plotted against the average of the results obtained by the two methods (x-axis). The mean of the differences ± SD of bias is -0.05 ± 0.40, n = 21.

	Discussion

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We present an assay design for the quantification of mRNA that is suitable for routine use. An important feature of this user-friendly assay is that it is based on a calibration curve, thereby allowing samples to be accepted or rejected according to standard quality-control rules.

Traditional competitive RT-PCR techniques for quantification of gene expression requires coamplification of target mRNA and several different internal standard concentrations to determine the concentration of a specific mRNA in an unknown sample. This makes the method labor-intensive, time-consuming, and expensive if multiple samples are being analyzed. Our CURT-PCR assay makes it possible to avoid these difficulties and to improve the precision of the quantification.

To establish the CURT-PCR, we developed a method for the quantitation of EGFR mRNA, using RT-PCR with a heterologous RNA as an internal standard. The RNA internal standard is coamplified with EGFR mRNA in the sample with the same primer set. The primers were chosen in a region of complete homology between human, rat, and mouse EGFR, making it possible to quantitate EGFR mRNA in all three species. The synthetic RNA standard controls the efficiency of both the reverse transcription reaction and the PCR reaction. Tube-to-tube variation is thereby controlled with the RNA internal standard. The difference in size between standard and target EGFR mRNA allows separation of the corresponding amplification products by electrophoresis (16).

A mandatory requirement for the accurate quantitative RT-PCR is identical reverse transcription and PCR efficiencies of the RNA internal standard and target mRNA in the samples (1)(2)(19). In some studies, DNA internal standards have been used in quantitative RT-PCR (1)(17)(20), but it is important to mention that a DNA standard does not take into account the variability of the reverse transcription step, which is an important source of variability in cDNA synthesis and which has been reported to range from 5% to 90% (1)(2)(21). In addition, because the cDNA yield in the reverse transcription step is generally less than the theoretical value of 100%, the amount of sample RNA measured based on a DNA internal standard may well be underestimated (6). Furthermore, the efficiency of amplification between single- and double-stranded DNA templates may vary (22). When these factors are taken together, the RNA internal standard has the advantage of compensating for any variations in the efficiency of both reverse transcription and the subsequent PCR amplification.

The use of DNA or RNA internal standards with a deletion or insertion for competitive RT-PCR has been reported as an advantage when compared with standards with heterologous sequence (1)(5)(23)(24)(25), because sequence homology between the target and the internal standard is believed to be a prerequisite for equal amplification efficiency of the two molecules. However, such a strategy presents the possibility of heteroduplex formation, yielding DNA molecules containing one target strand and one internal-standard strand at high numbers of PCR cycles. The formation of heteroduplex molecules has been considered a disadvantage in accurate quantification (19)(26)(27). By use of a nonhomologous internal standard, we eliminated heteroduplex formation during competitive RT-PCR, and we demonstrate that the heterologous RNA standard used in our study and EGFR mRNA in the sample have equal amplification efficiency. This suggests that it is not necessary to have the same nucleotide sequence in the target and RNA internal standard to obtain equal RT-PCR amplification efficiency. This is supported by others who have demonstrated that amplification efficiency is mainly determined by primer binding (2)(4)(23)(27). The use of in vitro synthesized RNA internal standard in RT-PCR appears to be a feasible way to quantitate expression from a specific gene, avoiding laborious cloning techniques.

Despite its enormous utility in molecular biology, many technical obstacles remain with the use of RT-PCR to quantitate mRNA. A major advantage of the procedure we present is the use of a calibration curve combined with the use of an RNA internal standard instead of a DNA internal standard and an internal standard with a heterologous rather than homologous sequence. This design is likely to give an accurate assay that has considerably lower imprecision than previous assays. It is also important that the assay is easy to perform and more cost-effective than the conventional competitive RT-PCR. If, for example, 10 samples are to be analyzed, one will need 55 tubes to run a competitive RT-PCR (5 for each sample and 5 for the control sample), but only 17 tubes are needed to run CURT-PCR (6 for the calibration curve and 1 for each sample, including the control sample). Usually, the maximal number of samples we run in a single round of competitive RT-PCR is 10. With CURT-PCR, we can run ~40 samples using the same amount of reagents and time.

Recently, Tsai and Wiltbank (28) reported a standard curve-based RT-PCR method that quantifies each sample by comparison with a standard curve that is produced with different amounts of synthetically generated native RNA and a constant amount of competitor RNA. In our study, we used a pool of mRNA to generate the calibration curve, and we demonstrated that the amplification efficiency of the internal standard and sample RNA is identical.

In conclusion, CURT-PCR improves and simplifies quantification of gene expression. CURT-PCR has several advantages over traditional methods because it is less time-consuming, requires less total RNA, is cheaper to perform, has lower imprecision, and allows samples to be accepted or rejected on the basis of established rules for quality assurance. The newly developed assay seems well suited to quantitate the expression of EGFR in clinical samples, such as tumor biopsies.

	Acknowledgments

 
This work was supported by the Danish Medical Research Council, the Danish Cancer Society, and the Danish Cancer Research Foundation. We acknowledge the excellent technical assistance of Birgit West Mortensen and Alice Villemoes.

	Footnotes

 
¹ Nonstandard abbreviations: RT-PCR, reverse transcriptase-PCR; EGFR, epidermal growth factor receptor; and CURT-PCR, calibrated user-friendly reverse transcriptase polymerase chain reaction.

	References

Top Abstract Introduction Material and Methods Results Discussion References

 

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