- Email: [email protected]
Escherichia coli DNA Contamination in AmpliTaq Gold Polymerase Interferes with TaqMan Analysis of lacZ
ARTICLE
doi:10.1006/mthe.2002.0548, available online at http://www.idealibrary.com on IDEAL
Escherichia coli DNA Contamination in AmpliTaq Gold Polymerase Interferes with TaqMan Analysis of lacZ Jonna K. Koponen,1,* Anna-Mari Turunen,1,* and Seppo Ylä-Herttuala1,2,† 1
A. I. Virtanen Institute, 2Gene Therapy Unit and Department of Medicine, University of Kuopio, FIN-7021, Kuopio, Finland *These authors contributed equally to this work.
†
To whom correspondence and reprint requests should be addressed. Fax: +358-17-163751. E-mail: [email protected].
Real-time PCR is a powerful method for the quantification of gene expression in biological samples. This method uses TaqMan chemistry based on the 5⬘-exonuclease activity of the AmpliTaq Gold DNA polymerase which releases fluorescence from hybridized probes during synthesis of each new PCR product. Many gene therapy studies use lacZ, encoding Escherichia coli -galactosidase, as a marker gene. Our results demonstrate that E. coli DNA contamination in AmpliTaq Gold polymerase interferes with TaqMan analysis of lacZ gene expression and decreases sensitivity of the method below the level required for biodistribution and long-term gene expression studies. In biodistribution analyses the contamination can lead to false-negative results by masking low-level lacZ expression in target and ectopic tissues, and false-positive results if sufficient controls are not used. We conclude that, to get reliable TaqMan results with lacZ, adequate controls should be included in each run to rule out contamination from AmpliTaq Gold polymerase. Key Words: real-time PCR, TaqMan, lacZ
INTRODUCTION
RESULTS
Real-time PCR can be used for the quantification of gene expression accurately and reproducibly over a wide dynamic range [1,2]. After reverse transcription of the RNA sample, we use kinetic PCR technology with ABI PRISM 7700 Sequence Detection system and fluorogenic probes. TaqMan probes are labeled with a reporter dye on the 5⬘ end and a quencher dye on the 3⬘ end. TaqMan chemistry is based on the 5⬘-exonuclease activity of the AmpliTaq Gold DNA polymerase [3], which releases fluorescence from hybridized probes during synthesis of each new PCR product. The amount of fluorescence is monitored throughout the course of the PCR allowing for real-time detection and quantification. In gene therapy, real-time PCR can be used for the evaluation of biodistribution, time courses, and pharmacokinetics of the transgenes and vectors [4,5]. A widely used marker gene for gene transfer applications is lacZ, encoding Escherichia coli -galactosidase [6]. The usefulness of lacZ is due to the easy detection of -galactosidase activity in cells and tissue samples with the X-gal staining method [7]. Our aim was to optimize the TaqMan method to quantify the expression of lacZ after different gene transfer procedures, for example with adenoviral and lentiviral vectors. That turned out to be problematic because of the significant background amplification of lacZ signal from AmpliTaq Gold polymerase.
Figure 1 shows typical amplification plots obtained with the first primer pair and probe. As a positive sample, 1 l of cDNA sample corresponding to 10 ng reverse transcribed total RNA was used (+SAMPLE). Controls included no template control (NTC) with water instead of the template, no amplification control (NAC) without reverse primer in the amplification mix, and no reverse transcription control (–RT) where MultiScribe enzyme was omitted in the reverse transcription step. For the positive sample the cycle number where the fluorescence passed the fixed threshold level, called threshold cycle (CT) was 27. For NTC CT = 33 and for –RT CT = 32. An amplification plot was not generated for NAC. Similar results were obtained reproducibly with different product lots of AmpliTaq Gold and also with the second primer pair and probe (data not shown). Amplification seen in NTC was due to E. coli genomic DNA contamination in the AmpliTaq polymerase. Withdrawal of AmpErase uracil N-glycosylase had no effect on CT of NTC and no signal was seen in NTC for 18S ribosomal RNA (data not shown). The possibility that a contamination in RT-enzyme MultiScribe caused false results was excluded by the –RT control. Gene transfer efficiency in many target tissues of gene therapy remains quite low. Thus, high CT values for marker
220
AND
DISCUSSION
MOLECULAR THERAPY Vol. 5, No. 3, March 2002 Copyright © The American Society of Gene Therapy 1525-0016/02 $35.00
doi:10.1006/mthe.2002.0548, available online at http://www.idealibrary.com on IDEAL
ARTICLE
FIG. 1. Amplification plots from lacZ real-time PCR. +SAMPLE (duplicates in blue and pink) indicates amplification plots from a known positive sample, RT (duplicates in orange and yellow) indicates amplification plots from a sample where reverse transcriptase enzyme was omitted, and NTC (duplicates in red and green) indicates amplification plots from the amplification mix without template. Representative results are shown from TaqMan analyses repeated several times with different lots of AmpliTaq Gold DNA polymerase.
gene expression are expected, especially in biodistribution and time course studies. According to our experience, fluorescence in NTC passed the threshold as early as cycle number 33. It cannot simply be considered as background amplification because amplification plots passing threshold at later cycles are very likely in biodistribution analyses [8]. In fact, real-time PCR has been used for biodistribution analyses [4,5]. When lacZ results have been reported, it is possible that some of the findings may be have been interfered with by the contamination , because copy numbers reported from various tissues were several times lower than the copy numbers in the lowest standards used [4]. According to our experience, at least 40 cycles should be used in TaqMan assays to detect low amounts of vector distribution. However, exponential amplification in NTC is repeatedly present after 40 cycles with lacZ as a target. We conclude that false-positive lacZ expression in realtime PCR quantification using AmpliTaq Gold polymerase should be carefully controlled when the amplicon is homologous to E. coli lacZ. The results also underline the need to develop ultrapure reagents for quantitative realtime PCR reactions.
MATERIALS
AND
METHODS
RNA for the optimization was obtained from the PA317 cell line with stable expression of lacZ [7,9]. Total RNA was extracted using Qiagen RNeasy Mini Kit (Qiagen, Valencia, CA) and treated with DNaseI (Promega, Madison, WI). RNA was reverse transcribed with TaqMan Gold RT-PCR kit (PE Biosystems, Foster City, CA). RT-reaction mix (10 l) consisted of 1⫻ TaqMan RT buffer, 5.5 mM MgCl2, 500 M of each dNTPs, 2.5 M random hexamers, 0.4 U/l RNase inhibitor, 1.25 U/l MultiScribe Reverse Transcriptase, and 100 ng of total RNA. Conditions for reverse transcription were 10 minutes at 25⬚C, 30 minutes at 48⬚C, and 5 minutes at 95⬚C. In real-time PCR two different combinations of primers and probes were tested. The primer and probe sequences were designed with Primer Express software version 1.0 (PE Biosystems). In the first combination a
MOLECULAR THERAPY Vol. 5, No. 3, March 2002 Copyright © The American Society of Gene Therapy
70-bp fragment was amplified (nt 235–305; GenBank acc. no. V00296) with the following primers: forward, 5⬘-CCTGAGGCCGATACTGTCGT-3⬘; reverse, 5⬘-TTGGTGTAGATGGGCGCAT-3⬘; probe, 5⬘-TCCCCTCAAACTGGCAGATGCACG-3⬘. In the second combination of primers the length of amplicon was 65 bp (nt 65–130; GenBank acc. no. V00296) with the following primers: forward, 5⬘-CCCAACTTAATCGCCTTGCA-3⬘; reverse, 5⬘GCGGGCCTCTTCGCTATT-3⬘; probe, 5⬘-CACATCCCCCTTTCGCCAGCTG3⬘. Both TaqMan probes contained a fluorescent reporter, dye 6-carboxyfluorescein (FAM), covalently linked to the 5⬘ end of the oligonucleotide and a quencher dye, 6-carboxytetramethylrhodamine (TAMRA), attached to the 3⬘ end via a linker group (PE Biosystems). We used 18S ribosomal RNA as an endogenous amplification control. Amplification of endogenous control was done in different wells than the amplification of lacZ (singleplex). Primers and probes were obtained from TaqMan Ribosomal RNA Control Reagents (PE Biosystems). After the RT step, real-time PCRs were performed in MicroAmp Optical 96-well Reaction Plates with Optical Caps (PE Biosystems) by use of the ABI PRISM 7700 Sequence Detection System (PE Biosystems). Each 50 l reaction consisted of 1⫻ TaqMan Universal PCR Master Mix (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 10 mM EDTA, 60 nM passive reference dye (6-carboxyX-rhodamine), 0.2 mM dATP, 0.2 mM dCTP, 0.2 mM dGTP, 0.2 mM dUTP, 5.5 mM MgCl2, 8% glycerol, 1.25 U AmpliTaq Gold DNA polymerase, and 0.5 U AmpErase uracil N-glycosylase), 300 nM forward primer, 300 nM reverse primer, 100 nM TaqMan probe, and varying amount of template. TaqMan PCR Core Reagent Kit (PE Biosystems) was also used to prepare the PCR master mixes with similar content. The following controls were included in every run: no template control (NTC) with water instead of the template; no reverse transcription control (–RT) where MultiScribe enzyme was omitted in the RT step; and no amplification control (NAC) without reverse primer in the amplification mix. PCR mixes were prepared in a laminar hood to avoid any contamination. Every sample was analyzed in duplicate. Cycling parameters were first, the uracil-N-glycosylase (UNG) reaction at 50⬚C for 2 minutes, then AmpliTaq Gold activation at 95⬚C for 10 minutes, followed by 40 cycles of denaturation at 95⬚C for 15 seconds, and combined annealing and extension at 60⬚C for 1 minute. Emitted fluorescence from each well was measured during both the denaturation and annealing/extension steps in every cycle. Amplification plots were constructed using the ABI PRISM 7700 Sequence Detection System software, version 1.7 (PE Biosystems).
ACKNOWLEDGMENTS This study was supported by grants from Finnish Academy and Sigrid Juselius Foundation.
221
ARTICLE
doi:10.1006/mthe.2002.0548, available online at http://www.idealibrary.com on IDEAL
RECEIVED FOR PUBLICATION OCTOBER 11, 2001; ACCEPTED JANUARY 18, 2002.
REFERENCES 1. Heid, C. A., Stevens, J., Livak, K. J., and Williams, P. M. (1996). Real time quantitative PCR. Genome Res. 6: 986–994. 2. Gibson, U. E., Heid, C. A., and Williams, P. M. (1996). A novel method for real time quantitative RT-PCR. Genome Res. 6: 995–1001. 3. Holland, P. M., Abramson, R. D., Watson, R., and Gelfand, D. H. (1991). Detection of specific polymerase chain reaction product by utilizing the 5⬘-3⬘ exonuclease activity of Thermus aquaticus DNA polymerase. Proc. Natl. Acad. Sci. USA 88: 7276–7280. 4. Senoo, M., et al. (2000). Adenovirus-mediated in utero gene transfer in mice and guinea pigs: tissue distribution of recombinant adenovirus determined by quantitative TaqMan-polymerase chain reaction assay. Mol. Genet. Metab. 69: 269–276,
222
doi:10.1006/mgme. 2000.2984. 5. Hackett, N. R., et al. (2000). Use of quantitative TaqMan real-time PCR to track the time-dependent distribution of gene transfer vectors in vivo. Mol. Ther. 2: 649–656, doi:10.1006/mthe.2000.0203. 6. Kalnins, A., Ruther, O. K., and Muller-Hill, B. (1983). Sequence of the lacZ gene of Escherichia coli. EMBO J. 2: 593–597. 7. Ylä-Herttuala, S., et al. (1995). Transfer of 15-Lipoxygenase gene into rabbit iliac arteries results in the appearance of oxidation-specific lipid-protein adducts characteristic of oxidized low density lipoprotein. J. Clin. Invest. 95: 2692–2698. 8. Hiltunen, M. O., et al (2000). Biodistribution of adenoviral vector to nontarget tissues after local in vivo gene transfer to arterial wall using intravascular and periadventitial gene delivery methods. Faseb J. 14: 2230–2236. 9. Mann, R., Mulligan, R. C., and Baltimore, D. (1983). Construction of a retrovirus packaging mutant and its use to produce helper-free defective retrovirus. Cell 33: 153–159.
MOLECULAR THERAPY Vol. 5, No. 3, March 2002 Copyright © The American Society of Gene Therapy
doi:10.1006/mthe.2002.0548, available online at http://www.idealibrary.com on IDEAL
Escherichia coli DNA Contamination in AmpliTaq Gold Polymerase Interferes with TaqMan Analysis of lacZ Jonna K. Koponen,1,* Anna-Mari Turunen,1,* and Seppo Ylä-Herttuala1,2,† 1
A. I. Virtanen Institute, 2Gene Therapy Unit and Department of Medicine, University of Kuopio, FIN-7021, Kuopio, Finland *These authors contributed equally to this work.
†
To whom correspondence and reprint requests should be addressed. Fax: +358-17-163751. E-mail: [email protected].
Real-time PCR is a powerful method for the quantification of gene expression in biological samples. This method uses TaqMan chemistry based on the 5⬘-exonuclease activity of the AmpliTaq Gold DNA polymerase which releases fluorescence from hybridized probes during synthesis of each new PCR product. Many gene therapy studies use lacZ, encoding Escherichia coli -galactosidase, as a marker gene. Our results demonstrate that E. coli DNA contamination in AmpliTaq Gold polymerase interferes with TaqMan analysis of lacZ gene expression and decreases sensitivity of the method below the level required for biodistribution and long-term gene expression studies. In biodistribution analyses the contamination can lead to false-negative results by masking low-level lacZ expression in target and ectopic tissues, and false-positive results if sufficient controls are not used. We conclude that, to get reliable TaqMan results with lacZ, adequate controls should be included in each run to rule out contamination from AmpliTaq Gold polymerase. Key Words: real-time PCR, TaqMan, lacZ
INTRODUCTION
RESULTS
Real-time PCR can be used for the quantification of gene expression accurately and reproducibly over a wide dynamic range [1,2]. After reverse transcription of the RNA sample, we use kinetic PCR technology with ABI PRISM 7700 Sequence Detection system and fluorogenic probes. TaqMan probes are labeled with a reporter dye on the 5⬘ end and a quencher dye on the 3⬘ end. TaqMan chemistry is based on the 5⬘-exonuclease activity of the AmpliTaq Gold DNA polymerase [3], which releases fluorescence from hybridized probes during synthesis of each new PCR product. The amount of fluorescence is monitored throughout the course of the PCR allowing for real-time detection and quantification. In gene therapy, real-time PCR can be used for the evaluation of biodistribution, time courses, and pharmacokinetics of the transgenes and vectors [4,5]. A widely used marker gene for gene transfer applications is lacZ, encoding Escherichia coli -galactosidase [6]. The usefulness of lacZ is due to the easy detection of -galactosidase activity in cells and tissue samples with the X-gal staining method [7]. Our aim was to optimize the TaqMan method to quantify the expression of lacZ after different gene transfer procedures, for example with adenoviral and lentiviral vectors. That turned out to be problematic because of the significant background amplification of lacZ signal from AmpliTaq Gold polymerase.
Figure 1 shows typical amplification plots obtained with the first primer pair and probe. As a positive sample, 1 l of cDNA sample corresponding to 10 ng reverse transcribed total RNA was used (+SAMPLE). Controls included no template control (NTC) with water instead of the template, no amplification control (NAC) without reverse primer in the amplification mix, and no reverse transcription control (–RT) where MultiScribe enzyme was omitted in the reverse transcription step. For the positive sample the cycle number where the fluorescence passed the fixed threshold level, called threshold cycle (CT) was 27. For NTC CT = 33 and for –RT CT = 32. An amplification plot was not generated for NAC. Similar results were obtained reproducibly with different product lots of AmpliTaq Gold and also with the second primer pair and probe (data not shown). Amplification seen in NTC was due to E. coli genomic DNA contamination in the AmpliTaq polymerase. Withdrawal of AmpErase uracil N-glycosylase had no effect on CT of NTC and no signal was seen in NTC for 18S ribosomal RNA (data not shown). The possibility that a contamination in RT-enzyme MultiScribe caused false results was excluded by the –RT control. Gene transfer efficiency in many target tissues of gene therapy remains quite low. Thus, high CT values for marker
220
AND
DISCUSSION
MOLECULAR THERAPY Vol. 5, No. 3, March 2002 Copyright © The American Society of Gene Therapy 1525-0016/02 $35.00
doi:10.1006/mthe.2002.0548, available online at http://www.idealibrary.com on IDEAL
ARTICLE
FIG. 1. Amplification plots from lacZ real-time PCR. +SAMPLE (duplicates in blue and pink) indicates amplification plots from a known positive sample, RT (duplicates in orange and yellow) indicates amplification plots from a sample where reverse transcriptase enzyme was omitted, and NTC (duplicates in red and green) indicates amplification plots from the amplification mix without template. Representative results are shown from TaqMan analyses repeated several times with different lots of AmpliTaq Gold DNA polymerase.
gene expression are expected, especially in biodistribution and time course studies. According to our experience, fluorescence in NTC passed the threshold as early as cycle number 33. It cannot simply be considered as background amplification because amplification plots passing threshold at later cycles are very likely in biodistribution analyses [8]. In fact, real-time PCR has been used for biodistribution analyses [4,5]. When lacZ results have been reported, it is possible that some of the findings may be have been interfered with by the contamination , because copy numbers reported from various tissues were several times lower than the copy numbers in the lowest standards used [4]. According to our experience, at least 40 cycles should be used in TaqMan assays to detect low amounts of vector distribution. However, exponential amplification in NTC is repeatedly present after 40 cycles with lacZ as a target. We conclude that false-positive lacZ expression in realtime PCR quantification using AmpliTaq Gold polymerase should be carefully controlled when the amplicon is homologous to E. coli lacZ. The results also underline the need to develop ultrapure reagents for quantitative realtime PCR reactions.
MATERIALS
AND
METHODS
RNA for the optimization was obtained from the PA317 cell line with stable expression of lacZ [7,9]. Total RNA was extracted using Qiagen RNeasy Mini Kit (Qiagen, Valencia, CA) and treated with DNaseI (Promega, Madison, WI). RNA was reverse transcribed with TaqMan Gold RT-PCR kit (PE Biosystems, Foster City, CA). RT-reaction mix (10 l) consisted of 1⫻ TaqMan RT buffer, 5.5 mM MgCl2, 500 M of each dNTPs, 2.5 M random hexamers, 0.4 U/l RNase inhibitor, 1.25 U/l MultiScribe Reverse Transcriptase, and 100 ng of total RNA. Conditions for reverse transcription were 10 minutes at 25⬚C, 30 minutes at 48⬚C, and 5 minutes at 95⬚C. In real-time PCR two different combinations of primers and probes were tested. The primer and probe sequences were designed with Primer Express software version 1.0 (PE Biosystems). In the first combination a
MOLECULAR THERAPY Vol. 5, No. 3, March 2002 Copyright © The American Society of Gene Therapy
70-bp fragment was amplified (nt 235–305; GenBank acc. no. V00296) with the following primers: forward, 5⬘-CCTGAGGCCGATACTGTCGT-3⬘; reverse, 5⬘-TTGGTGTAGATGGGCGCAT-3⬘; probe, 5⬘-TCCCCTCAAACTGGCAGATGCACG-3⬘. In the second combination of primers the length of amplicon was 65 bp (nt 65–130; GenBank acc. no. V00296) with the following primers: forward, 5⬘-CCCAACTTAATCGCCTTGCA-3⬘; reverse, 5⬘GCGGGCCTCTTCGCTATT-3⬘; probe, 5⬘-CACATCCCCCTTTCGCCAGCTG3⬘. Both TaqMan probes contained a fluorescent reporter, dye 6-carboxyfluorescein (FAM), covalently linked to the 5⬘ end of the oligonucleotide and a quencher dye, 6-carboxytetramethylrhodamine (TAMRA), attached to the 3⬘ end via a linker group (PE Biosystems). We used 18S ribosomal RNA as an endogenous amplification control. Amplification of endogenous control was done in different wells than the amplification of lacZ (singleplex). Primers and probes were obtained from TaqMan Ribosomal RNA Control Reagents (PE Biosystems). After the RT step, real-time PCRs were performed in MicroAmp Optical 96-well Reaction Plates with Optical Caps (PE Biosystems) by use of the ABI PRISM 7700 Sequence Detection System (PE Biosystems). Each 50 l reaction consisted of 1⫻ TaqMan Universal PCR Master Mix (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 10 mM EDTA, 60 nM passive reference dye (6-carboxyX-rhodamine), 0.2 mM dATP, 0.2 mM dCTP, 0.2 mM dGTP, 0.2 mM dUTP, 5.5 mM MgCl2, 8% glycerol, 1.25 U AmpliTaq Gold DNA polymerase, and 0.5 U AmpErase uracil N-glycosylase), 300 nM forward primer, 300 nM reverse primer, 100 nM TaqMan probe, and varying amount of template. TaqMan PCR Core Reagent Kit (PE Biosystems) was also used to prepare the PCR master mixes with similar content. The following controls were included in every run: no template control (NTC) with water instead of the template; no reverse transcription control (–RT) where MultiScribe enzyme was omitted in the RT step; and no amplification control (NAC) without reverse primer in the amplification mix. PCR mixes were prepared in a laminar hood to avoid any contamination. Every sample was analyzed in duplicate. Cycling parameters were first, the uracil-N-glycosylase (UNG) reaction at 50⬚C for 2 minutes, then AmpliTaq Gold activation at 95⬚C for 10 minutes, followed by 40 cycles of denaturation at 95⬚C for 15 seconds, and combined annealing and extension at 60⬚C for 1 minute. Emitted fluorescence from each well was measured during both the denaturation and annealing/extension steps in every cycle. Amplification plots were constructed using the ABI PRISM 7700 Sequence Detection System software, version 1.7 (PE Biosystems).
ACKNOWLEDGMENTS This study was supported by grants from Finnish Academy and Sigrid Juselius Foundation.
221
ARTICLE
doi:10.1006/mthe.2002.0548, available online at http://www.idealibrary.com on IDEAL
RECEIVED FOR PUBLICATION OCTOBER 11, 2001; ACCEPTED JANUARY 18, 2002.
REFERENCES 1. Heid, C. A., Stevens, J., Livak, K. J., and Williams, P. M. (1996). Real time quantitative PCR. Genome Res. 6: 986–994. 2. Gibson, U. E., Heid, C. A., and Williams, P. M. (1996). A novel method for real time quantitative RT-PCR. Genome Res. 6: 995–1001. 3. Holland, P. M., Abramson, R. D., Watson, R., and Gelfand, D. H. (1991). Detection of specific polymerase chain reaction product by utilizing the 5⬘-3⬘ exonuclease activity of Thermus aquaticus DNA polymerase. Proc. Natl. Acad. Sci. USA 88: 7276–7280. 4. Senoo, M., et al. (2000). Adenovirus-mediated in utero gene transfer in mice and guinea pigs: tissue distribution of recombinant adenovirus determined by quantitative TaqMan-polymerase chain reaction assay. Mol. Genet. Metab. 69: 269–276,
222
doi:10.1006/mgme. 2000.2984. 5. Hackett, N. R., et al. (2000). Use of quantitative TaqMan real-time PCR to track the time-dependent distribution of gene transfer vectors in vivo. Mol. Ther. 2: 649–656, doi:10.1006/mthe.2000.0203. 6. Kalnins, A., Ruther, O. K., and Muller-Hill, B. (1983). Sequence of the lacZ gene of Escherichia coli. EMBO J. 2: 593–597. 7. Ylä-Herttuala, S., et al. (1995). Transfer of 15-Lipoxygenase gene into rabbit iliac arteries results in the appearance of oxidation-specific lipid-protein adducts characteristic of oxidized low density lipoprotein. J. Clin. Invest. 95: 2692–2698. 8. Hiltunen, M. O., et al (2000). Biodistribution of adenoviral vector to nontarget tissues after local in vivo gene transfer to arterial wall using intravascular and periadventitial gene delivery methods. Faseb J. 14: 2230–2236. 9. Mann, R., Mulligan, R. C., and Baltimore, D. (1983). Construction of a retrovirus packaging mutant and its use to produce helper-free defective retrovirus. Cell 33: 153–159.
MOLECULAR THERAPY Vol. 5, No. 3, March 2002 Copyright © The American Society of Gene Therapy