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Bioconjugate Chem. 2002, 13, 172−176
Electrochemical Detection of Gene Expression in Tumor Samples: Overexpression of Rak Nuclear Tyrosine Kinase Paul M. Armistead and H. Holden Thorp* Department of Chemistry and Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-3290. Received December 13, 2000; Revised Manuscript Received March 2, 2001
Absolute quantification of Rak nuclear tyrosine kinase mRNA in breast tissue samples was determined by competitive RT-PCR. The total RNA from the same samples was also chemically amplified through conventional RT-PCR, and the relative amounts of these amplified RT-PCR products were determined by adsorption onto an indium tin oxide (ITO) electrode followed by electrochemical detection. The electrochemical detection was performed using the inorganic metal complex Ru(bpy)32+ (bpy ) 2,2′ bipyridine) to catalyze the oxidation of the guanine residues of the immobilized RT-PCR products. Using the competitive RT-PCR values as standards, it was found that an optimized conventional RTPCR coupled with electrochemical detection provides a simple method for measuring relative gene expression among a series of mRNA samples from breast tumors. The use of electrochemical detection potentially eliminates the need for gel electrophoresis and fluorescent or radioactive labels in detecting the target genes.
Abnormalities in the expression of specific genes have been linked to a large and increasing number of diseases (1-7). The identification of overexpressed genes provides a promising framework for identifying diagnostics as well as protein or small molecule drugs; this approach has been particularly successful in studies of HER2-neu in breast cancer (8-10). Several methods are currently available to quantify mRNA expression in biological samples; these methods typically provide semiquantitative information in some whole genome expression assays and absolute quantification in some single gene assays (11-16). Easily automated methods for detecting relative gene expression would allow for routine use of gene expression in diagnostics and drug discovery (17, 18). Our laboratory has developed a method for detecting trace nucleic acids based on electrocatalysis with cyclic voltammetric detection (19-21). In these experiments, DNA molecules are adsorbed onto indium tin oxide (ITO) electrodes with a surface area of 12.6 mm2, placed in an electrochemical cell containing 10 µM of the electrontransfer mediator Ru(bpy)32+ (bpy ) 2,2′-bipyridine), and detected via catalytic oxidation of the guanine base according to the following reactions (22):
Ru(bpy)32+ f Ru(bpy)33+ + e-
(1)
Ru(bpy)33+ + guanine f Ru(bpy)32+ + guanine+ (2) The resulting cyclic voltammmogram shows a dramatic enhancement in the Ru(bpy)32+ oxidation wave as the generated Ru(bpy)33+ is recycled to Ru(bpy)32+ by electrons from the guanine bases. Experiments with model PCR products showed that the sensitivity of this system was 44 amol/mm2 of electrode area, which translates to a detection limit for the 12.6 mm2 electrodes of roughly 550 amol (23). A number of other electrochemical techniques are under development, and the advantages of electrochemical approaches have been discussed in detail (24-29). In this application, the electrochemical tech-
nique can be used to replace gel electrophoresis performed following the RT-PCR protocol. The electrochemical technique could ultimately be performed in a miniaturized electrode array for analysis in high throughput without fluorescent or radioactive labels. We report here that our electrochemical technique can be used to determine the mRNA expression level of Rak nuclear tyrosine kinase in a series of breast tissue samples. Rak (also called Frk) is a distant relative in the Src family of tyrosine kinases and is overexpressed in roughly 30% of human epithelial (breast and colon) cancers (30). The absolute quantity of Rak mRNA measured by competitive RT-PCR (14) ranged from 440 to 2300 zmol/µg of total RNA in breast tissues, which are typical values for the mRNA of signaling molecules (16). The same samples were analyzed by conventional RTPCR with electrochemical detection (23), and a strong correlation was observed between the electrochemical current and the absolute quantity of mRNA determined by competitive RT-PCR. EXPERIMENTAL SECTION
Materials and Reagents. All oligonucleotides were synthesized at the Lineberger Comprehensive Cancer Center Nucleic Acid Core Facility at the University of North Carolina at Chapel Hill. Tissue Samples were from the Tissue Procurement Facility at UNC-CH. Of the seven tissue samples studied, sample 35A was normal tissue, sample 54B was a primary tumor with no signs of invasion through the basal lamina, and the others were invasive breast tumors. Sample 35B was an invasive breast tumor from the same patient as sample 35A. BT474 cells were a gift from the lab of Dr. Bill Cance. All water used was in-house distilled water that was further purified on a Milli-Q water purification system. Water for use in RNA manipulations was further treated with diethyl pyrocarbonate, DEPC, (Sigma, St. Louis, MO). N,N-Dimethyl formamide, sodium phosphate, sodium acetate, and sodium chloride were from Mallinckrodt
10.1021/bc000129y CCC: $22.00 © 2002 American Chemical Society Published on Web 02/06/2002
Electrochemical Detection of Gene Expression in Tumors Scheme 1
(Paris, KY). Ru(bpy)3Cl2 was purchased from Aldrich (Milwaukee, WI) and purified by recrystallization from acetonitrile. ITO electrodes were purchased from Delta Technologies (Stillwater, MN). Total RNA from both cell culture and tissue samples was purified on Qiagen RNeasy columns according to the manufacturer’s directions. All electrochemical measurements were performed in a one-compartment cell on an Princeton Applied Research (Princeton, NJ) model 273A potentiostat that was controlled by a Pentium computer. An Ag/AgCl reference electrode was used (Cypress Systems, Lawrence, KS), and a platinum wire was used as the auxiliary electrode. Fluorescent staining of DNA in agarose was performed using SYBR-green (Molecular Probes, Eugene, OR). Fluorescently stained gels were scanned on a Molecular Dynamics (San Jose, CA) Storm 860 phosphorimager using the blue fluorescence mode. Scanned gels were analyzed using ImageQuaNT software. Competitive RT-PCR. Synthesis of an appropriate Rak mRNA competitor followed the strategy outlined in Scheme 1 (15). A Rak RT-PCR product was produced that contained the T7 promoter sequence as well as a “loopout” region. The reverse transcription was carried out as follows. A 14-µL reaction mix consisting of 1 µg of total BT-474 RNA, 714 µM dNTPs (Amersham), and 0.4 µg/ µL random hexamers (Life Technologies) was prepared. The mix was heated to 75 °C for 3 min and immediately cooled on ice. To the mix was added 4 µL of 5× buffer (Life Technologies), 1 µL of RNasin (Promega), and 1 µL (200 U) of M-MLV reverse transcriptase (Life Technologies). The reaction was allowed to proceed for 1 h at 42 °C and then stopped by heating the mix to 90 °C for 10 min. A 50-µL PCR was made that contained 5 µL of the reverse transcription mix in 1× buffer, 2.5 mM MgCl2, 125 µM dNTPs, 250 nM forward primer (TAA TAC GAC TCA CTC TAG GGC CTA TCT GGA GTC TCG GAA GCA GAT TTT GGA CTT GCC AGA), 250 nM reverse primer (GGG CAC CTG TCA TAC CAC TGT), and 1 U Taq polymerase (Life Technologies). The reaction was performed with 1 cycle of 95 °C for 5 min followed by 30 cycles of 94 °C for 20 s, 62 °C for 30 s, 72 °C for 40 s, and 1 cycle 72 °C for 5 min. The purity of the product was determined by agarose gel electrophoresis. The gel revealed a single band at the appropriate size (250 bp). The PCR product was purified on Qiagen Qiaquick PCR purification columns. In vitro transcription was performed by assembling a 20-µL reaction mix that contained 40 mM Tris (pH ) 7.5), 10 mM MgCl2, 10 mM DTT, 8 mM NTPs, 50 µg/mL BSA,
Bioconjugate Chem., Vol. 13, No. 2, 2002 173
170 nM PCR template, and 1× T7 polymerase that was a gift from the lab of Dr. Kevin Weeks. The reaction was allowed to proceed for 7 h at 37 °C. Following the reaction, 1 µL of DNase (Ambion) was added to the reaction and allowed to incubate for 30 min. To the reaction, were added 15 µL of 5 M ammonium acetate, 15 µL of 100 mM sodium EDTA, and 100 µL of DEPC water. The reaction was extracted with acid phenol/ chloroform/isoamyl alcohol. The organic phase was backextracted with 50 µL of DEPC water. The 200 µL of aqueous mix was precipitated with ice cold 2-propanol overnight. The RNA pellet was resuspended in TE buffer and run on a 4% denaturing polyacrylamide gel. The transcription products were visualized by UV shadowing, and the single visible product was cut from the gel using a clean razor blade. The gel fragment was placed into 300 µL of eluting solution containing 500 mM ammonium acetate, 1 mM EDTA, and 0.2% SDS (Ambion). The gel slice was kept in the solution overnight at 4 °C. The eluting solution was transferred from the gel slice, and the eluted RNA was ethanol precipitated overnight revealing a glassy pellet. The pellet was resuspended in TE buffer. The amount of product, 1.8 µg (23 pmol), was determined by UV-vis spectroscopy at 260 nm. Competitive RT-PCR was performed by preparing 6 reverse transcription mixes for each tissue sample. A 15µL reaction mix was prepared that contained 0.5 µg of total RNA, a known quantity of the competitor (either 31.3, 62.5, 125, 250, 500, or 1000 zmol), 667 µM dNTPs, and 667 nM reverse primer. The mixes were heated and cooled on ice as described above. To each mix was added 4 µL of 5× buffer and 1 µL (200 U) of M-MLV reverse transcriptase. The reverse transcription and PCR were performed as described above except a different forward primer (GGC CTA TCT GGA GTC TCG GAA) was used. Each reaction mix was run on a 2% agarose gel and stained with SYBR-Green for 20 min. Staining with SYBR-Green was performed after the electrophoresis to minimize measurement errors due to dye migration in the gel. The stained gel was scanned with a Molecular Dynamics Storm 860 phosphorimager in the blue fluorescence mode. The band intensities resulting from both the native Rak mRNA amplification (296 bp) and the competitor RNA amplification (232 bp) were measured, and background fluorescence was subtracted. For each tissue sample, the logarithm of the competitor signal/ native signal in each mix was plotted vs the logarithm of the amount of competitor added for each mix. The best linear fit was calculated, and the antilogarithm of x at y ) 0 was calculated. This value yielded the absolute quantity of Rak mRNA in the tissue sample. Conventional RT-PCR Amplification of Rak. RTPCR of Rak was performed on all tissue samples in parallel, except sample 55B, which did not yield enough RNA. Six reverse transcription reactions were assembled (1 for each sample). Each 15-µL reaction contained 0.5 µg of total RNA, 0.4 µg/µL random hexamers, and 667 µM dNTPs. The random hexamers and dNTPs were originally pooled into a master mix and then pipetted into each individual reverse transcription mix. The mixes were heated and cooled as described above. Four microliters of 5× buffer and 1 µL (200 U) of M-MLV were added. These reagents were premixed to ensure that equivalent amounts of enzyme were added to each reverse transcription reaction. The reactions were again performed at 42 °C for 1 h and were stopped by heating to 90 °C for 10 min. PCR was assembled for each tissue sample as a 100µL solution that contained 10 µL of reverse transcription
174 Bioconjugate Chem., Vol. 13, No. 2, 2002
mix, 1× buffer, 2.5 mM MgCl2, 125 µM dNTPs, 250 nM forward primer (GGC CTA TCT GGA GTC TCG GAA), 250 nM reverse primer (GGG CAC CTG TCA TAC CAC TGT), and 2 U Taq polymerase. The reagents were premixed except the reverse transcription mix. Ninety microliters of the master mix was added to 10 µL of the reverse transcription mix. This step again ensured that equivalent amounts of Taq were added to each reaction. The reaction proceeded as follows: 1 cycle of 95 °C for 5 min followed by 28 cycles of 94 °C for 20 s, 62 °C for 30s, 72 °C for 40 s, and 1 cycle 72 °C for 5 min. Five microliters of each product were analyzed by agarose gel electrophoresis. Purification of Rak RT-PCR Products. After RTPCR, 90 µL of each PCR were purified on Qiagen Qiaquick PCR purification columns. The purification was performed according to the manufacturer’s instructions, except for the final elution step. At this step, each column was treated with 30 µL of 10 mM sodium acetate pH ) 8.0 (pH raised by addition of sodium hydroxide). The columns were allowed to sit for one minute and were then centrifuged at 14 000 rpm for 2 min. Again, 5 µL of each product was analyzed by agarose gel electrophoresis. The pre- and post-purification gels were used to confirm that the relative amounts of RT-PCR among samples did not change significantly after purification. Electrochemical Detection of Rak RT-PCR Products. ITO electrodes were cleaned using the protocol of Willit and Bowden (31). The electrodes were cleaned by sonication in 2-propanol for 15 min, and twice in Milli-Q water each time for 15 min. The electrodes were air-dried. For each electrode, 5 µL of purified PCR product were added to 45 µL of DMF. The DMF mixtures were pipetted onto the electrodes, which were placed in a constant humidity chamber for 30 min (23). The electrodes were then washed in Milli-Q water for 3 min on a rotary mixer and allowed to air-dry. The DNA modified electrodes were placed in a onecompartment electrochemical cell. The cell contained 150 µL of 10 µM Ru(bpy)32+ in 50 mM sodium phosphate buffer at pH ) 7.0. Electrochemical detection was performed using cyclic voltammetry at a scan rate of 10 V s-1. The potential range was 0-1300 mV. All measurements were made relative to an Ag/AgCl reference electrode. Because of the large charging currents generated at this fast scan rate, background subtraction was performed. The background scans were taken with no Ru(bpy)32+ at ITO electrodes that had been treated with a 9:1 DMF/sodium acetate solution (without DNA) and washed as above. The background electrodes were placed in the same electrochemical cell, and cyclic voltammetry was performed only in the presence of 50 mM sodium phosphate. Immobilized DNA was quantitated by comparison of catalytic currents for DNA-modified electrodes scanned with Ru(bpy)32+ compared to the currents obtained for Ru(bpy)32+ only on electrodes not modified with DNA. RESULTS AND DISCUSSION
Absolute Quantification of Rak mRNA in Breast Tissue Samples. Competitive RT-PCR was performed on the seven breast tissue samples and BT-474 cells following a procedure adapted from published protocols (14). A typical agarose gel of a competitive RT-PCR is shown in Figure 1 (top). Quantities of amplified material and internal standard were determined by fluorescence and plotted as in Figure 1 (bottom). The linear fits from all of the analyses had an average R-value of 0.99 ( 0.01
Armistead and Thorp
Figure 1. Results of competitive RT-PCR for sample 35B. (A) Agarose gel showing that as the amount of competitor added to each reaction is increased, the ratio of the amount of native RTPCR product (296 bp) decreases relative to the amount of competitor RT-PCR product (232 bp). (B) The logarithm of the band intensity ratio versus the logarithm of the quantity of competitor.
Figure 2. Quantity of Rak mRNA from competitive RT-PCR given as zmol of Rak mRNA per µg of total RNA for the specified sample. Quantities determined for each sample as in Figure 1.
and an average slope of 0.98 ( 0.14. Observation of slopes near unity indicates that the standard RNA is behaving as a proper competitor (32). The competitive RT-PCR results for the seven breast tissues and BT-474 cells are shown in Figure 2. Rak mRNA expression ranged from 440 zmol/µg of total RNA in normal breast tissue (sample 35A) to 2300 zmol/µg of total RNA in sample 2049B. A greater than 2-fold increase in expression (compared to normal sample 35A) was observed in two of the six breast cancer samples, which is consistent with the finding of Cance et al. that Rak is overexpressed in roughly 30% of breast tumors (30). The six samples examined here were for the purposes of demonstrating the electrochemical method and not meant to reflect a statistically relevant population; nevertheless, the determined quantities and trends are entirely consistent with the biological expectations. While Rak overexpression is clearly evident, it should be
Electrochemical Detection of Gene Expression in Tumors
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Figure 4. Plot of the absolute quantity of Rak mRNA in the series of breast tumor samples (Figure 2) versus the peak current from the corresponding cyclic voltammograms (Figure 3). Note that the error bars for each axis are given in Figures 2 and 3.
Figure 3. (A) Cyclic voltammograms for ITO electrodes with no DNA or modified with the RT-PCR products from six breast tissue samples at a scan rate of 10 V/s with 10 µM Ru(bpy)32+ in 50 mM phosphate buffer (pH 7). (B) Peak current for each sample.
noted that the magnitude of overexpression is relatively mild, expecially when compared to other oncogenes such as HER-2, which can vary in expression over 3 orders of magnitude (16, 33). Electrochemical Quantification of Rak RT-PCR Products. For electrochemical analysis, conventional RT-PCR was performed on each tissue sample, and the resulting PCR reactions were purified to remove the unreacted nucleotides and pyrophosphate, which inhibit adsorption of the DNA onto the electrode (23). Agarose gel electrophoresis was performed on all of the samples both before and after the purification step to ensure that purification did not affect the relative quantities of the Rak PCR products. After purification, the PCR products were adsorbed to the electrodes using a procedure similar to that we have published previously (23). Electrochemical detection of the Rak RT-PCR products was performed by cyclic voltammetry in the presence of Ru(bpy)32+. We have shown previously that the peak currents obtained by this method increase with increasing amounts of adsorbed PCR product (23). We have also determined previously that a scan rate of 10 V/s and a 10 µM concentration of Ru(bpy)32+ provide the optimal sensitivity (23). Here we apply this method to clinical samples for the first time. Representative cyclic voltammograms of Ru(bpy)32+ at unmodified electrodes (no DNA) and at ITO electrodes modified with the Rak RTPCR products from the breast tissue samples are shown in Figure 3 along with a bar graph giving the peak currents and associated errors. Figure 4 shows the correlation between the peak currents determined by cyclic voltammetry and the absolute quantity of mRNA applied to the electrode determined by competitive RT-PCR. A good correlation is observed between the expression level and the electrochemical signal. The signal appears to maximize somewhat for the higher expression levels. We have
shown previously that the electrochemical method exhibits a high dynamic range for PCR amplicons applied to electrodes and directly quantitated by radiolabeling (23); quantities of immobilized PCR products spanning 2 orders of magnitude give distinct electrochemical signals. Thus, the saturation in Figure 4 at high copy numbers is likely due to the reduced dynamic range of the conventional RT-PCR compared to that of competitive RT-PCR (14) and not to a lower dynamic range of the electrochemical method as compared to fluorescence detection. The accuracy of the electrochemical method is therefore more than sufficient to distinguish changes in the quantities of conventional RT-PCR products. In particular, the two samples indicating overexpression (2028B and 2049B) were identified using both methods. CONCLUSIONS
Rak mRNA is expressed in the range of 100-2300 zmol/µg of total RNA in seven breast tissue samples and BT-474 cells as determined by competitive RT-PCR. In this group, 2 of 6 breast tumors expressed Rak at a level more than twice that of normal breast tissue (sample 35A). This result supports the findings of Cance and Liu that Rak is overexpressed in roughly 30% of all invasive breast tumors (30). A semiquantitative conventional RTPCR approach coupled with electrochemical detection led to similar Rak expression data. The two breast tumors with the highest absolute Rak mRNA quantity also gave the largest electrochemical signals (which were also 2× greater than the electrochemical signal from sample 35A). The magnitude of the differences in expression from sample to sample was diminished in the conventional RTPCR versus the competitive RT-PCR measurements, probably as a result of the reduced dynamic range of the conventional method. Catalytic electrochemical detection of RT-PCR products offers a rapid method for determining the relative amount of a specific mRNA in a series of biological samples. The adsorption of DNA to the ITO electrodes is complete in 30 min, and performing the cyclic voltammogram takes less than 1 s. LITERATURE CITED (1) Andreelli, F., Laville, M., Ducluzeau, P. H., Vega, N., Vallier, P., Khalfallah, Y., Riou, J. P., and Vidal, H. (1999) Defective regulation of phosphatidylinositol-3-kinase gene expression in skeletal muscle and adipose tissue of noninsulin-dependent diabetes mellitus patients. Diabetologia 42, 358-364. (2) Callahan, L. M., Vaules, W. A., and Coleman, P. D. (1999) Quantitative decrease in syaptophysin message expression and increase in cathepsin D message expression in Alzheimer
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