Abstract
Background: Rapid-cycling, real-time PCR instruments bring the opportunity for improved intraoperative detection of metastasis to sentinel lymph nodes. Rapid, standardized, and internally controlled assays need to be developed that are sensitive and accurate.
Methods: We describe rapid, multiplexed, internally controlled, quantitative reverse transcription-PCR (QRT-PCR) assays for tyrosinase and carcinoembryonic antigen mRNAs on the SmartCycler (Cepheid). We used a temperature-controlled primer-limiting approach to eliminate amplification of the endogenous control gene as soon as its signal had reached threshold. Positive-control oligonucleotide mimics were incorporated into all reactions to differentiate failed reactions from true negative samples.
Results: The optimized assays for rapid QRT-PCR yielded results with threshold cycle values that were only 1–2 cycles higher than slower, more conventional protocols. In rapid PCR, the temperature-controlled multiplex assay was quantitative over a dynamic range of at least 15 cycles, compared with only 6 cycles for conventional multiplexing methods. All histologically positive lymph nodes examined were also QRT-PCR positive for the appropriate marker, and the exogenous, internal positive-control mimics produced signals in all negative samples.
Conclusion: Internally controlled, rapid QRT-PCR assays can be performed in an intraoperative time frame and with sufficient sensitivity to detect histologically identified metastases to lymph nodes.
Despite the enormous power and flexibility of PCR, this technology has been slow to find its way into clinical diagnostic laboratories. In large part, this is because the PCR process (from sample processing to reaction set-up and data analysis) is labor-intensive, prone to contamination, and technically quite demanding. As a result, false-positive and -negative results are frequent and constitute a major concern in the clinical setting. Nevertheless, most major clinical diagnostic laboratories continue to perform a handful of internally validated, PCR-based assays for various applications, such as viral detection (1)(2) and minimal residual disease detection in leukemia patients (3)(4)(5). Technical advances that simplify and, if possible, automate PCR procedures would greatly enhance the reproducibility and reliability of these assays.
One such technical advance, the introduction of rapid cycling, quantitative PCR instruments, is opening up new potential uses for molecular testing. The ability to complete PCR assays in <30 min now makes it possible to perform time-dependent, point-of-care molecular diagnostics, such as testing for group B streptococcus in pregnant women (6) and nosocomial agents in immunocompromised patients (7). In cancer diagnostics, such rapid tests bring the possibility of primary cancer diagnosis on core biopsies or fine-needle aspirates while the patient is still in the clinic, tumor profiling at the time of surgery to determine response to chemotherapy, and intraoperative testing of lymph nodes and surgical margins to determine the extent of disease and treatment options. For example, reverse transcription-PCR (RT-PCR)1 has been shown in many studies to improve the sensitivity of cancer cell detection in lymph nodes of cancer patients otherwise staged as node negative (8)(9)(10)(11). These patients are at higher risk for disease recurrence and may benefit from more aggressive therapy. In the case of lung cancer, for example, patients with mediastinal lymph node involvement may benefit from neoadjuvant chemotherapy, but lymph node status needs to be determined before major surgical intervention (12)(13)(14). This can be achieved through the use of minimally invasive surgical staging or ultrasound-guided fine-needle aspirates (15). Accurate intraoperative lymph node diagnosis would allow node-negative patients to undergo complete tumor resection, whereas surgery would be halted in node-positive patients to allow administration of chemotherapy.
A similar scenario exists for patients with breast cancer or melanoma. In both diseases, the treatment for sentinel lymph node-positive patients is complete lymph node dissection. Because current intraoperative lymph node analysis methods are not very sensitive [∼70% in breast cancer (16) and 47% in melanoma (17)], many patients are not diagnosed as lymph node positive until after their initial surgery. These patients then have to undergo a second surgical procedure to complete the lymph node dissection. A more sensitive, intraoperative lymph node assessment would clearly benefit these patients. This example illustrates just one potential use of rapid PCR-based assays, but many more are likely to be forthcoming in the near future. Once again, however, advances are needed in PCR technology to make these exciting possibilities practical in the clinical diagnostic laboratory.
The recent introduction of fluorescence-based PCR and RT-PCR has greatly simplified data analysis steps by eliminating the need for post-PCR processing and gel electrophoresis. Added benefits of this technology include reduced assay contamination (because PCR tubes are never opened) and, of course, the ability to obtain highly accurate and reproducible quantitative results. Quantification not only enhances the clinical utility of many potential diagnostic tests (8)(18)(19), but also provides the ability to verify assay sensitivity and reproducibility from test to test with the use of external quality-control standards (8). Internal quality controls are also required, including amplification of endogenous control sequences to check for quality and quantity of the template DNA or RNA, and internal positive controls (IPCs) to verify that the assay worked in the case of a negative result (20)(21). Although “internal” controls can be set up in separate tubes, it would be advantageous if all assays could be run (multiplexed) in the same PCR tube, taking advantage of differently colored fluorogenic probes to distinguish the PCR products. Unfortunately, multiplex PCR does not work well when the amplification targets are not present in similar abundance at the beginning of the reaction, and it is especially difficult to maintain accurate quantification over a wide range of target concentrations (22). The dynamic range of a quantitative multiplex assay can be improved to some degree by severely limiting the PCR primer concentration for the more abundant gene in the PCR reaction. Although this approach works reasonably well for standard RT-PCR assays, the concept of primer limiting goes against the requirements of a rapid PCR assay, where higher primer concentrations are necessary to maintain amplification. We describe a novel method for multiplex PCR with internal controls for performance in rapid quantitative RT-PCR (QRT-PCR) assays. In addition, we apply this method to detect tumor cells in lymph nodes.
Materials and Methods
tissues and rna isolation
All patient tissue samples were obtained through University of Pittsburgh Institutional Review Board-approved protocols. Lymph nodes from patients with esophageal cancer and benign nodes obtained incidentally from patients undergoing antireflux procedures were snap-frozen in liquid nitrogen, and RNA was extracted using the RNeasy® Mini Kit (Qiagen) according to the manufacturer’s protocol. The histologically positive and negative lymph nodes from melanoma patients were obtained as formalin-fixed, paraffin-embedded archived samples, and RNA was extracted using previously described protocols (23). Cells from A549 lung adenocarcinoma cell line [with low-level expression of carcinoembryonic antigen (CEA)] were generously provided by Dr. Michael Epperly (Department of Radiation Oncology, University of Pittsburgh, Pittsburgh, PA).
development of the rapid pcr assay
Rapid PCR was initially tested using cDNA synthesized from A549 RNA mixed with RNA from a lymph node that was histologically positive for metastatic esophageal cancer. PCR primers and probes for β-glucuronidase (β-GUS) and CEA were designed to be cDNA specific with amplicon sizes of 81 and 77 bp, respectively. Known pseudogenes for CEA were avoided, and a control PCR on 200 ng of genomic DNA was negative. PCR was performed in 25-μL reactions containing 800 nM each primer, 200 nM each probe (primer and probe sequences are shown in Table 1⇓ ), and the PCR master mixture [1× Platinum Taq reaction buffer; 4.5 mM MgCl2, 300 μM each deoxynucleotide triphosphate, and 0.09 U/μL Platinum Taq DNA polymerase (Invitrogen)]. Assays were performed on the SmartCycler® (Cepheid, Sunnyvale, CA) and analyzed with SmartCycler software (Ver. 1.2b). PCR denaturation (95 °C) times of 10, 7, 5, 2, and 1 s (annealing/extension time was 30 s) and annealing/extension (64 °C) times of 30, 15, 10, 7, 6, and 3 s were initially tested on a single cDNA input. Because the optics require a minimum of 6 s to read, the data for the extension time trial were obtained by reading the fluorescence during the denaturation phase (10-s hold). This allowed us to directly compare the data for the 3-s extension with longer extension times. The optimal rapid PCR cycling conditions (1-s denaturation followed by a 6-s annealing/extension) were then used to compare the rapid PCR protocol with more conventional cycling times of denaturation for 10 s and annealing/extension for 30 s. This was carried out with a serially diluted CEA calibrator to determine any effect of the rapid PCR protocol over a wide range of CEA input. The CEA cDNA [reverse-transcribed using human colon total RNA (Ambion) as template] was serially diluted (eightfold dilutions; each dilution had a volume ≥ 600 μL) in a constant background of β-GUS template (purified β-GUS PCR product) in amounts that were empirically determined to give constant β-GUS cycle threshold (Ct) values between 19 and 20 cycles. Similar experiments were carried out to optimize and validate the tyrosinase assay.
Primer and probe sequences.
development of the rapid reverse transcription assay
The RNA input for the QRT-PCR was a mixture of 12.5 ng of spleen RNA (Ambion) and 0.66 ng from a primary lung adenocarcinoma. QRT-PCR reactions were performed for β-GUS and CEA mRNA in 25 μL containing the PCR master mixture described above plus 60 nM each of β-GUS and CEA reverse transcription primers (per 25-μL volume), 1 μL of SensiScript reverse transcriptase (Qiagen), and 0.8 units/μL RNase inhibitor. Reverse transcription was carried out at 48 °C in a 20-μL volume, and the PCR primers and probes were added to the reaction mixture in a 5-μL volume during a 70 °C hold after the reverse transcription. This was done to maintain PCR specificity and sensitivity (24). Reverse transcription times of 30, 10, 7, 5, 3, and 2 min were tested to determine the fastest reverse transcription possible without loss of sensitivity. The PCR times were held constant at 1 s for denaturation and 6 s for annealing/extension.
multiplex pcr assays
In the temperature-controlled multiplex PCR, both primer and probe sets for CEA and β-GUS were added to the reaction mixture. In this case, however, the β-GUS primers (β-GUS80; 400 nM) were redesigned to have a calculated melting temperature (Tm) of 50 °C compared with the CEA primers, which have a calculated Tm of 60 °C. PCR was performed with a 1-s denaturation, a 4-s hold at 53 °C for annealing, and a 6-s hold at 64 °C for extension and optical reading (total annealing/extension time, 10 s). The 53 °C annealing step was eliminated one cycle after the β-GUS amplification plot reached threshold (determined beforehand in a separate singleplex reaction), and subsequent cycles were carried out with only the 64 °C annealing/extension step as in the optimal, rapid PCR singleplex assay.
Simple multiplexing was performed using 800 nM primers for both genes and used our standard β-GUS81 (Tm = 60 °C) primer set. The conventional primer-limited multiplex assay was carried out using 300 nM β-GUS81 primers because this was the lowest concentration that did not produce a significant increase (more than one cycle) in the β-GUS Ct values in a rapid PCR. For the conventional methodologies, the first 20 PCR cycles were performed with an annealing/extension time of 10 s at 64 °C followed by a change to 6 s for the remaining cycles. This was done to better simulate the conditions used in the temperature-controlled multiplex assay and to allow direct comparison of the methods. All three multiplex methods were tested on serial dilutions of CEA cDNA (as described above) to determine the accuracy and dynamic range compared with singleplex reactions.
IPCs
The IPCs were DNA oligonucleotides that had the same primer sequences as the target gene (CEA or tyrosinase) but a different internal probe sequence (Table 2⇓ ). Selected sites in the IPCs were synthesized with uracil instead of thymine so that contamination with the highly concentrated mimic could be controlled by use of uracil DNA glycosylase if required. The IPCs were added to the reaction master mixture in amounts that were determined empirically to give Ct values of 36–37 cycles. The rapid triplex assays were then performed as described for the primer-limited multiplex (duplex) assay above, with the addition of the IPC probe at a concentration of 200 nM.
CEA and tyrosinase IPC mimics.1
lymph node assays
Histologically positive and negative lymph nodes from patients with esophageal cancer and melanoma were tested using rapid, multiplexed QRT-PCR to demonstrate the utility of this method. Lymph node RNA was analyzed using a triplex QRT-PCR (β-GUS, CEA, or tyrosinase and appropriate mimic) with the temperature-controlled primer-limiting technique. Reverse transcription was carried out as described above with a 5-min reverse transcription step followed by a 30-s hold at 70 °C for addition of PCR primers. Ct values for β-GUS were determined in a prior, singleplex assay so that the temperature change could be initiated at the appropriate cycle in the multiplex reaction.
Results
rapid pcr development
Rapid PCR assays were developed separately for all genes in singleplex assays before they were used in a multiplex reaction. For development of the rapid PCR assays, we evaluated the effect of reducing both the denaturation time and the annealing/extension time to determine any observed changes in Ct values. For the denaturation time test, the annealing/extension time was kept constant at 30 s, and for the annealing/extension time test, the denaturation time was kept constant at 10 s. We found that reducing the denaturation time to 1 s had no effect on the Ct values for either the CEA or β-GUS genes (Fig. 1A⇓ ). Similarly, reducing the annealing/extension time had minimal effect (∼0.7 cycles) on the Ct values for both genes down to 6 s, whereas a 3-s annealing/extension step increased the Ct by 1.22 cycles for CEA and 1.92 cycles for β-GUS compared with the 30-s annealing/extension control (Fig. 1B⇓ ). Thus, for further testing we chose to use a 1-s denaturation and a 6-s annealing/extension step.
Optimization of the rapid QRT-PCR assay for β-GUS and CEA.
Panel A compares the Ct values for assays with different denaturation times and a 30-s extension time. Panel B compares the effect of different extension times on the Ct value when the denaturation time is held constant at 10 s. Panel C demonstrates the effect of different reverse transcription times when the PCR conditions are constant. Error bars, 95% confidence intervals.
To evaluate the effect of the rapid PCR assay on sensitivity and quantification over a wide range of CEA input amounts, we generated a calibration curve using serial dilutions of CEA cDNA in a constant background of β-GUS PCR target. Using this calibration curve, we compared the rapid assay with a more conventional PCR with 10-s denaturation and 30-s annealing/extension steps (Fig. 2⇓ ). The Ct values for CEA and β-GUS were increased by an average of 1.6 and 0.2 cycles, respectively, in the rapid assay. These increases appeared to be consistent from point to point, and as a result, the quantitative ability of the rapid assay was equal to that of the slower PCR. The total PCR time to run 40 cycles was 15 min for the rapid assay vs 48 min for the slower PCR protocol.
Comparison of Ct values for β-GUS and CEA assayed by slow conventional PCR and rapid PCR.
Values are for eightfold serial dilutions of CEA in a constant background of β-GUS. Slow conventional PCR (10-s denaturation followed by a 30-s annealing/extension step): ⋄, β-GUS; ▴, CEA. Rapid PCR (1-s denaturation followed by a 6-s annealing/extension step): □, β-GUS; ▦, CEA.
rapid reverse transcription
Rapid analysis of gene expression by QRT-PCR requires that both the reverse transcription and PCR components of the assay are rapid. For this reason, we evaluated the effect of reducing the reverse transcription time from 30 min down to 2 min (Fig. 1C⇑ ). The results showed increases in Ct value of only 0.44 and 1.9 cycles for β-GUS and CEA, respectively, with a 5-min reverse transcription and 0.48 and 3.23 cycles, respectively, with a 2-min reverse transcription, compared with 30 min. Although none of these differences were significant for β-GUS, there did appear to be a trend toward increased Ct with reduced time for CEA. However, despite the slight loss in sensitivity for CEA relative to the 30-min reverse transcription, there was no significant difference between a 10-min reverse transcription and the 3- or 5-min reverse transcription.
The combined effect of decreasing both the reverse transcription and PCR times on the sensitivity of the assay was evaluated using four different reverse transcription and PCR time combinations (Fig. 3⇓ ). Again a trend was observed toward higher Ct values with shorter RT-PCR protocols, but with a 20-min total RT-PCR time (5-min reverse transcription and PCR with a 1-s extension and a 6-s denaturation), the Ct difference was only 1.4 cycles for both genes compared with a 38-min total RT-PCR (10-min reverse transcription and PCR with a 10-s extension and a 15-s denaturation).
Comparison of Ct values for QRT-PCR assays with various overall run times.
♦, β-GUS; ▪, CEA. Run times: 15 min (2-min reverse transcription followed by PCR with a 1-s denaturation and a 3-s extension), 17 min (2-min reverse transcription followed by PCR with a 1-s denaturation and a 6-s extension), 20 min (5-min reverse transcription followed by PCR with a 1-s denaturation and a 6-s extension), and 38 min (10-min reverse transcription followed by PCR with a 5-s denaturation and a 15-s extension). Error bars, 95% confidence intervals.
multiplex pcr assay
We tested different multiplexing methods, using the same dilution series of CEA in a constant background of β-GUS that was used for the rapid PCR development. This dilution series spanned ∼12 cycles, corresponding to a 4096-fold difference in the starting CEA abundance between the first and the last points on the curve. This dilution series was used to compare singleplex reactions for each gene [Fig. 1A⇑ in the data supplement that accompanies the online version of this article (available athttp://www.clinchem.org/content/vol48/issue8)] with our temperature-controlled primer-limited multiplex PCR (Fig. 1B⇑ in the data supplement), a simple multiplex PCR (Fig. 1C⇑ in the data supplement), and a conventional primer-limited multiplex PCR (Fig. 1D⇑ in the data supplement). When a simple multiplex reaction was carried out for both CEA and β-GUS, only the first three of five points from the series reached threshold for CEA before 40 cycles, whereas the reaction for the more abundant β-GUS amplified consistently (not shown). Similar results were observed for the conventional primer-limited reaction, whereas the temperature-controlled primer-limiting method produced results almost identical to the singleplex reactions.
Perhaps more relevant than the range of CEA concentrations is the range of ΔCt (difference in Ct value between the more abundant β-GUS gene and the CEA target gene) that can be achieved with the different methods. In these experiments, the β-GUS Ct was constant for all assays, between 19 and 20 cycles; thus the largest ΔCt in this dilution series with a singleplex assay was almost 15 cycles (∼32 000-fold difference in abundance). Fig. 4⇓ shows the ΔCt values plotted for each of the three multiplexing methods as well as the singleplex data. In the simple multiplex reaction, the third point in the series had a Ct value of 38.5 cycles compared with 29.0 for the same point in the singleplex reaction, whereas subsequent points failed to amplify at all. Thus, the simple multiplex reaction demonstrates a lack of quantification, with a true ΔCt of ∼8 cycles, as well as a very limited dynamic range. The conventional primer-limited multiplex reaction worked a little better (ΔCt for the third dilution point was 4.0 cycles higher than for the singleplex reaction), but once again, dilution points four and five failed to amplify. Thus, it seems that despite its feasibility in standard PCR reactions, conventional primer limiting is not amenable to rapid assays, in which increased primer concentrations are required. In comparison, the temperature-controlled multiplex reaction amplified all points on the dilution series, and no difference was observed in the ΔCt until the last point. These results clearly demonstrate that the temperature-controlled primer limit was comparable to singleplex reactions in the rapid quantitative PCR assay, whereas all other multiplexing approaches remained inadequate. Furthermore, we have used this technique on other targets, such as tyrosinase, and the method appears to be easily adaptable to new targets.
Comparison of the singleplex PCR (♦) for CEA with temperature-controlled multiplex (○), ordinary multiplex (□), and conventional primer-limiting multiplex (▴) PCR.
ΔCt values for a serial dilution of CEA in constant background of β-GUS are compared. A ΔCt of 25 denotes a dilution point that failed to amplify.
lymph node analysis using rapid, multiplex rt-pcr
For lymph node analysis, IPC mimics for CEA and tyrosinase were included in the multiplex reactions. This internally controlled, rapid RT-PCR multiplex assay was used to evaluate lymph nodes from patients with esophageal cancer and melanoma as well as benign lymph nodes from patients without cancer. In three histologically positive esophageal cancer lymph nodes, high CEA expression was detected. In lymph nodes from noncancer patients, two of five nodes had no detectable CEA expression, and of the remaining three, CEA expression on the highest expressing negative node was 40-fold lower than the positive node with the lowest CEA expression. Furthermore, in cases in which CEA signal was detected, the IPC was not amplified (Fig. 5A⇓ ). However, in CEA-negative samples, there was amplification of the CEA IPC, confirming that negative results were indeed attributable to the absence of CEA expression and not a failed CEA PCR (Fig. 5B⇓ ).
Representative amplification plots for samples expressing CEA (A) and tyrosinase (C) and samples not expressing CEA (B) and tyrosinase (D).
Panels A and B show fluorescence curves for β-GUS (+), CEA (⋄), and the CEA IPC (—). Panels C and D show fluorescence curves for β-GUS (⋄), tyrosinase (—), and the tyrosinase IPC (∗).
We obtained similar results for melanoma lymph nodes, using tyrosinase mRNA as the cancer marker. We tested five lymph nodes that were histologically positive for melanoma and five lymph nodes without any evidence of cancer. Tyrosinase was detected at high concentrations in all histologically positive nodes and at very low concentrations in only one of the five negative nodes (data not shown). In the positive nodes, the expression of tyrosinase was higher than that of β-GUS, and as a result, only the tyrosinase gene amplification was seen. In all five negative nodes, β-GUS and the IPC were amplified (Fig. 5, C and D⇑ ).
Discussion
The introduction of rapid cycling PCR instruments capable of real-time fluorescence-based quantification has opened the door for application of molecular diagnostic assays in clinical situations in which time is limited or a rapid result would be of psychological or physical benefit to the patient. One case in which rapid molecular assays may be advantageous is in intraoperative lymph node staging of malignancies such as melanoma, breast, and lung cancer. Conventional analysis of frozen tissue sections is very quick, ∼20 min in most institutions (25), but the sensitivity of this method for detecting small tumor foci is relatively low. Furthermore, many patients who are staged as lymph node negative by histologic examination suffer recurrence of their disease, probably as a result of occult disseminated tumor cells that were missed by routine examination. There is much evidence that RT-PCR, and particularly QRT-PCR, can detect these occult cells and identify patients at high risk for recurrence. If this information can be obtained intraoperatively, surgeons will be able to make more informed treatment decisions regarding the need for adjuvant therapy and extent of resection.
We have shown previously that it is feasible to carry out QRT-PCR in an intraoperative time frame and that this technique can be superior to frozen sectioning and, at least, comparable to formalin-fixed histology (19). In the present work, we have further tested and validated our rapid QRT-PCR protocols and show that this complete assay can be carried out in less than 20 min. Although this time frame is adequate for intraoperative testing, there are still several practical and technical hurdles that need to be addressed before rapid QRT-PCR can be considered feasible in the clinical diagnostic setting. We believe that the assays must be standardized to allow data comparison between sites in multicenter trials, automated (including RNA isolation) to eliminate operator-dependent variability and contamination, and controlled for all aspects of the assay, including internal endogenous and exogenous positive controls. The last of these requirements, quality controls, necessitates the incorporation of multiplexing into the rapid QRT-PCR. At least two control reactions are required, an endogenous control gene to verify the presence and adequate quantity and quality of RNA and an exogenous positive control to verify that the target gene QRT-PCR functioned with adequate sensitivity in samples that are negative for target gene expression. Together, these two controls ensure that a negative result is truly negative and not a result of failed RT-PCR. Thus, at least a triplex PCR reaction is needed.
Unfortunately, maintaining quantification in a multiplex PCR has not been easy even in a standard, slow PCR assay. The inhibition of the less abundant target sequence is probably a consequence of several factors, including the accumulation of pyrophosphate released from the addition of nucleotides during DNA synthesis and inhibition/sequestration of the Taq enzyme by the accumulating PCR product for the more abundant target (26). Initially, this inhibition is seen as a shift in the Ct value to a higher cycle number and a decrease in the total fluorescence when compared with a singleplex reaction (Fig. 1⇑ , panels A, C, and D, in the data supplement). If the difference in abundance of the two genes is too great, however, the less-abundant gene will fail to amplify completely. Thus, the multiplex reaction lacks quantification and suffers from a poor dynamic range. One way to overcome this inhibition is to stop amplification of the more abundant gene soon after signal has been detected and before pyrophosphate and PCR product have accumulated enough to cause inhibition. Conventional methods for this rely on limiting the primer concentration for the more abundant target gene such that the primers are used up, and PCR of that specific target stops, before inhibition can occur. Although this method works reasonably well in a slow PCR assay, reducing primer concentration in a rapid assay decreases PCR efficiency and, eventually, causes complete PCR failure. In our studies, we determined the lowest primer concentration that did not change the Ct value for β-GUS and tested this in a conventional primer-limited assay using our rapid PCR protocol. Although this was slightly better than multiplexing without a primer limit, quantification and the dynamic range were still poor compared with singleplex reactions.
Our novel method uses the same basic principle as conventional primer-limiting assays, but uses temperature to control the effective primer concentrations. In this method, the Tm of the primers for the more abundant species (β-GUS) are designed to function at a lower temperature than those for the target gene (CEA or tyrosinase). The PCR itself is carried out in two stages. The annealing step for the first stage of the PCR is performed at a relatively low temperature (53 °C in our example) until the amplification curve of the more abundant species reaches the detection threshold. The second stage of the PCR is then carried out with an annealing temperature that is at least 10 °C higher than the Tm of the low-temperature primers (64 °C for our example). The higher temperature of the second stage shifts the binding state of the low-temperature β-GUS PCR primers to favor the single-stranded state, effectively terminating amplification of β-GUS, whereas amplification of the CEA target continues uninhibited. Thus, we are able to maintain the high primer concentration needed for rapid PCR of the more abundant control gene and still maintain quantification of the target gene over a large dynamic range. Although a similar temperature-shifting approach (double stringency PCR) has been described previously to allow single-tube, nested PCR (27), it has not been used to terminate the PCR of a selected target after the point of detection, thus allowing a multiplex PCR to be performed for targets with large differences in abundance.
In addition to developing the rapid multiplexing technology, we have incorporated an IPC mimic that can be used to confirm that adequate RT-PCR sensitivity was achieved in samples negative for target gene expression. This mimic uses the same primers as the target gene and therefore controls for the function of these oligonucleotides. In the future, we anticipate replacing the DNA mimic with a RNA mimic that also includes the reverse transcription primer site. This will then control for both the reverse transcription and PCR steps of target gene amplification. By adding this mimic to samples at concentrations that are low enough to produce Ct values >36 cycles, we can be sure that RNA isolation and RT-PCR not only worked, but that they worked well enough to detect a very low abundance of target gene if it were present. In cases in which the target gene is highly expressed, the internal mimic fails to amplify, but this is acceptable because the mimic is intended only as a control for negative reactions.
In this and other studies, we have shown the potential use of rapid QRT-PCR for detection of metastatic tumor cells in the lymph nodes of cancer patients. Although this is a particularly exciting use of rapid molecular diagnostics, it is only one potential application. As new cancer markers are developed, it is possible that rapid QRT-PCR could also be used intraoperatively for analysis of surgical margins, in the clinic for analysis of biopsies or fine-needle aspirates, or for analysis of primary tumors to predict response to chemotherapy (28) or new biotherapies such as tyrosine kinase inhibitors (29)(30).
Although the dual-temperature PCR cycling required for the temperature-controlled multiplex reaction adds ∼2 min to the overall reaction time, a complete, multiplexed QRT-PCR assay can still be carried out in less than 22 min. With the addition of external cooling to the SmartCycler or its future generations, we believe that an extra 3–4 min can be saved. Also, in the present study, we predetermined the cycle number at which the temperature change needed to be initiated. Ultimately, a minor modification in the instrument’s software will automatically increase the PCR annealing temperature after the endogenous control gene fluorescence reaches threshold. Because all PCR sites are independently controlled, this will allow multiple reactions to be run at the same time with unknown amounts of starting RNA template. Finally, rapid QRT-PCR is still not feasible in a clinical setting unless RNA isolation can also be carried out very quickly and, preferably, in an automated process. To this end we are working with Cepheid to develop a fully integrated RNA isolation, reverse transcription, and quantitative PCR instrument (GeneXpert®) that will automate all of these processes and provide a QRT-PCR result in less than 30 min. The combination of our rapid, internally controlled multiplex technology with an automated sample-processing platform will allow for accurate and expeditious measurements in a closed environment. The simplicity and sensitivity of this integrated system should enable many new applications of molecular assays in cancer diagnostics.
Acknowledgments
This work was supported by NIH/NCI Research Grant CA 90665-01. Dr. Godfrey is currently a member of the scientific advisory board for Cepheid.
Footnotes
1 F, forward primer; R, reverse primer; FAM, 6-carboxyfluorescein; BHQ, Black Hole Quencher; RT, gene-specific primer for reverse transcription.
2 Dual-labeled fluorescent probe.
3 C and U were changed to their propyne pyrimidine counterparts and synthesized by Integrated DNA Technologies (Coralville, IA).
4 The GUS81 PCR primers were used for conventional primer limit as well as the simple multiplex and have the same Tm as the CEA and tyrosinase PCR primers.
5 The GUS80 PCR primers are shorter, lower Tm, primers that were utilized for the temperature-controlled multiplex PCR.
1 Italic, gene-specific primer sites; bold, probe sequence from bacterial β-galactosidase gene.
↵1 Nonstandard abbreviations: RT-PCR, reverse transcription-PCR; IPC, internal positive control; QRT-PCR, quantitative RT-PCR; CEA, carcinoembryonic antigen; β-GUS, β-glucuronidase; and Tm, melting temperature.
- © 2002 The American Association for Clinical Chemistry
References
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