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Polymerase Chain Reaction (PCR)-Based Construction of a Competitor Target for Quantitative Reverse Transcriptase PCR Measurement of Cytochrome P450 1A1 mRNA
280
NOTES & TIPS
IgG, alkaline phosphatase-conjugated BCIP/NBT as substrate (Sigma Chemical Co.)). In Fig. 1A the diffusion is very limited in comparison with the same reaction carried out directly on the plate (Fig. 1C). One of the advantages offered by this technique is the exact identification of the wild-type plaques which are to be avoided in further amplification. By this technique it is in fact possible to maintain under control the tendency of some clones to show occasionally the presence of b-galactosidase-positive plaques, a finding which may be the result of contamination with wild-type strains of the phage (presumably favored by a higher growth rate) or, less probable, by reversal to the wild type as a consequence of a precise excision at the cloning site of the cDNA insert. Perhaps the greatest advantage offered by the proposed technique concerns the possibility of running the control experiment in parallel with the traditional screening of the library in a very inexpensive way, in spare time without any need for extra cultures. We feel that it is always safer to have the experiment and the control carried out on the very same plate. REFERENCE 1. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Polymerase Chain Reaction (PCR)-Based Construction of a Competitor Target for Quantitative Reverse Transcriptase PCR Measurement of Cytochrome P450 1A1 mRNA Roland Fro¨tschl,* Ullrich Kleeberg,† Alfred G. Hildebrandt,† Ivar Roots,* and Ju¨rgen Brockmo¨ller* *Institute of Clinical Pharmacology, University Hospital Charite´, Humboldt University of Berlin, Schumannstrasse 20/21, D-10098 Berlin, Germany; and †Federal Institute of Drugs and Medical Products, Seestrasse 10, D-13353 Berlin, Germany Received March 26, 1996
Reliable quantification of mRNA by RT-PCR requires an internal or external standard cotranscribed and coamplified with the mRNA of interest. As internal standards provide only a relative quantification, external standards optimized for the assay of interest are required (1). Quantitative RT-PCR based on quantification of cDNA molecules transcribed from mRNA by using external DNA standards was already described ANALYTICAL BIOCHEMISTRY ARTICLE NO.
FIG. 1. PCR-mediated construction of artificial competitor mRNA. Steps of construction indicated as 1, 2, 3, and 4 and primers as well as gene and vector names and nucleotide positions are as described in the text.
by Gilliland et al. (2). However, different RT reaction efficiencies could not be excluded. Cytochrome P450 enzymes are important metabolizers of drugs, steroids, and xenobiotics, and as such they are of great medical interest (3). CYP1A1 is an important activator of polycyclic aromatic hydrocarbons and environmental pollutants (4), transforming such compounds into ultimate carcinogens (5). Therefore, it is of great clinical importance to evaluate the inducing potency of drugs and xenobiotics with high accuracy. Omeprazole is a known inducer of CYP 1A1 and 1A2 (6), but generally accepted systems to compare the inducing potency of omeprazole and related drugs (like the benzimidazolone domperidone) are still missing. Competitor artificial RNA (aRNA)1 was constructed from genomic CYP1A1, spanning the intron 6 from nucleotides 6435 to 6870 (7). After PCR of nucleotides 6435–6808, we distinguished between amplified contaminating genomic DNA and aRNA by an artificial EcoRI site introduced into the aRNA. The competitor aRNA was made in four steps (Fig. 1). Left and right arms were synthesized in two PCR (step 1), one with KON1(5*-ATCCTCTAGACCACCCGTTGCAGCAGGATAGCC-3*)/MU1 (5*-CCTACCTGAATTCTTTCTCACCCC-3*) and one with KON2 (5*-AAGTTCTGCAGGCTTTTACATCCCCAAGGGGCG-3*)/MU2 (5*-GGGGTGAGAAAGAATTCAGGTAGG-3*),each containing 1 mg of genomic DNA, 400 nM each primer, 80 mM each dNTP, 1.8 mM MgCl2 , 50 mM KCl, 10 mM Tris, pH 8.3, and 1 U of AmpliTaq DNA polymerase (Perkin–Elmer, Foster City, CA) in a final volume of 25 ml. Initial denaturation was 2 min at 957C, followed by 30 cycles with 1 min at 957C, 30 s at 527C, and 1 min at 727C and a final extension step at 727C for 7 min. PCR products were digested with EcoRI (Eurogen1
Abbreviations used: aRNA, artificial RNA; DMSO, dimethyl sulfoxide.
242, 280–282 (1996)
0468
0003-2697/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
10-18-96 11:54:42
abnt
AP: Anal Bio
281
NOTES & TIPS TABLE 1
Comparison of CYP1A1 mRNA Amounts in Hela Cells after Induction Inducer substance FIG. 2. RT-PCR of total RNA from Hela cells. One hundred nanograms of total RNA, DNAse digested, of omeprazole-induced (lanes 1 to 6) and domperidone-induced Hela cells (lanes 7 to 12). Serial dilutions of aRNA were added to the RT reactions: lanes 1 and 7, 40 fg; lanes 2 and 8, 8 fg; lanes 3 and 9, 1.6 fg; lanes 4 and 10, 0.32 fg; lanes 5 and 11, 0.064 fg; lanes 6 and 12, 0,0128 fg; lane M, 100-bp ladder.
Omeprazole Domperidone DMSO control
CYP1A1 mRNA CYP1A1 mRNA CYP1A1 mRNA per 100 ng molecules/100 ng molecules per total RNA total RNA cell 1.9 0.6 0.085
4600 1500
0.37 0.12
220
0.018
FIG. 3. Quantification of mRNA. Densitometer values of CYP1A1 mRNA-derived bands were plotted versus aRNA derived values. Curves result from linear regression.
gested with RNAse-free RQ1-DNAse (Promega) and quality was checked by gel electrophoresis. After purification on Ultrafree MC 30000 NMW filters, the amount of transcripts was quantitated by uv absorption at 260 nm. RT-reactions were set up according to the cDNA synthesis system manual of Life Technologies (Gaithersburg, MD), using MMLV-RT and a temperature-sensitive primer RT1 (5*-CAGGAAGAGAAAGAC-3*) (8). Reactions were adjusted to a final volume of 20 ml containing 8 U RNAsin (Promega) and 100 ng of total cellular RNA. Assays were overlaid with 25 ml mineral oil (Sigma, St. Louis, MO) and incubated at 377C for 1 h. Tubes were heated to 957C for 10 min and 30 ml of PCRmix was added at 957C, containing 50 mM KCl, 10 mM Tris, pH 8.3, 10 pmol of primers 1A8 (5*-GGCTTTTACATCCCCAAGGGGCG-3*) and 1A9 (5*-ATACACTTCCGCTTGCCCATGCC-3*), 0.5 mM MgCl2 , 67 mM each dNTP, and 1.25 U AmpliTaq. Cycling conditions were 1 min at 957C, 40 s at 667C, and 1 min 30 s at 727C for 30 cycles followed by 7 min at 727C. After EcoRI digestion the PCR products were separated on a 2% agarose gel. mRNA-derived amplificates show a 182-bp band, genomic DNA-derived amplificates a 373-bp band (7, 9), and artificial RNA-derived amplificates two bands of 288 and 85 bp. Equality of the 373- and 182-bp fragments in PCR amplification was ensured by amplifying both in one PCR and taking samples at different cycle numbers. Quotients of band intensities were the same at all cycles. Tests for intercalation of ethidium bromide showed that the same amounts of DNA of the different bands resulted in nearly equal densitometer values (data not shown). Serial dilutions of aRNA were added to a series of probes with constant RNA concentration and the resulting bands were recorded with a digital image system. Bands were integrated with the Quantiscan densitometry software from Biosoft, Cambridge, UK, and density values of 182-bp bands were corrected for size by addition of log a (a Å 288/182 Å 1.58) before correlation with 288-bp values. Concentration of mRNA was determined by linear regression analyses of aRNA intensities versus mRNA intensities.
AID
abnt
tec S.A., Seraing, Belgium) and purified. Left and right arms were ligated and the ligated fragment was amplified with primers KON1 and KON2 (step 2) as described above with an annealing temperature of 607C for 20 s. Five microliters of the PCR was digested with XbaI/PstI (Eurogentec), and the digested construct (now named 1A1Eco) was purified on Ultrafree MC 30000 NMW centrifugation filters (Millipore) and ligated into XbaI/PstI digested pT7T3 19U vector (Pharmacia, Uppsala, Sweden). The transcription target was amplified with primers pA1 (5*-GATTAAGTTGGGTAACGCCAGGG-3*) and pA2 (5*-TATGTTGTGTGGAATTGTGAGCG-3*) (step 3) as described above with an annealing step of 20 s at 607C. The amplificate was purified by agarose gel electrophoresis and filtration on Ultrafree MC units. The in vitro transcription (step 4) was carried out in a T7 in vitro transcription system from Promega (Madison, WI). Transcripts were di-
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282
NOTES & TIPS
Hela cells were cultured in DMEM, 10% (v/v) new born calf serum to near confluency and presumed inducers were added as 5 mM solutions in DMSO up to 50 mM final concentration of inducer. Controls were pretreated with the same amount of DMSO alone. Cells were incubated for 18 h at 377C in a 5% CO2 atmosphere and harvested by conventional trypsination, washed twice with Dulbecco’s PBS solution, and counted. Total RNA was isolated from cell pellets of 107 cells according to Chomczynski and Sacchi (10), digested with 10 U RNAse-free RQ1 DNAse (Promega) for 15 min at 377C, phenol–chloroform extracted, and ethanol precipitated. RNA concentration was determined spectrophotometrically and measured for 1A1 mRNA. Omitting the DNAse digestion resulted in contamination with genomic DNA, showing a 373-bp band in the RT-PCR assay. Results from induction tests of Hela cells with omeprazole and domperidone are demonstrated in Fig. 2. Fluorescence intensities of the CYP1A1 bands versus standard RNA bands were shown in Fig. 3. The number of standard RNA molecules of the intersection values of standard and mRNA curves was taken as the equilibrium value between mRNA molecules and standard molecules. Using serial dilutions with more narrow dilution steps (Fig. 3A) results in the same intersection values, demonstrating the high reliability and reproducibility of the measuring method. The described method is much more sensitive and absolutely selective compared to analysis by Northern blot or quantification of proteins with Western blot or enzyme-specific reporter reactions as already suggested by others (11, 12). Results of comparison of the intersection values in Fig. 3 are summarized in Table 1. Compared to uninduced controls we obtained a 22-fold increase in transcribed CYP1A1 mRNA for induction with omeprazole and a 7-fold induction with domperidone. The amount of 1A1 mRNA in omeprazole-induced Hela cells is about 4600 molecules or 1.9 fg per 100 ng of RNA. For approximately 2 to 5 ng of poly(A)/ mRNA out of 100 ng total cellular RNA, 1A1 mRNA is about 0.0001 to 0.00005%
AID
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total mRNA. In relation to the cell number used for RNA purification, the 1A1 mRNA level in uninduced Hela cells is about 1 molecule per 57 cells and increases in omeprazole-induced cells to 1 molecule per 2.7 cells and in domperidone-induced cells to 1 molecule per 8.3 cells. The increase in amount of CYP1A1 mRNA in Hela cells upon exposure to omeprazole or domperidone may be due to AH receptor-mediated transcriptional activation (13). The enzyme-inducing activity of domperidone is new and illustrates a possible application of our mRNA amplification method for drug development. The threefold lower induction may be due to lower affinity of benzimidazolones to the AH receptor compared to benzimidazoles and the differentiation of compounds according to their potency in enzyme induction may be of great medical relevance. Expression levels of CYP1A1 are considered to modulate the risk of cancer. REFERENCES 1. Chelly, J., and Kahn, A. (1994) in The Polymerase Chain Reaction (Mullis, K. B., Ferre´, F., and Gibbs, R. A., Eds.), pp. 97– 109, Birkhauser, Boston, MA. 2. Gilliland, G., Perrin, S., Blanchard, K., and Bunn, H. F. (1990) Proc. Natl. Acad. Sci. USA 87, 2725–2729. 3. Estabrook, R. W. (1990) Biochem. Soc. Trans. 18, 34–36. 4. Catteau, A., Douriez, E., Beaune, P., Poisson, N., Bonaiti-Pellie´, C., and Laurent, P. (1995) Pharmacogenetics 5, 110–119. 5. Beresford, A. P. (1993) Drug Metab. Rev. 25, 503–517. 6. Rost, K. L., Bro¨sicke, H., Brockmo¨ller, J., Scheffler, M., Helge, H., and Roots, I. (1992) Clin. Pharmacol. Ther. 52, 170–180. 7. Jaiswal, A. K., Gonzalez, F. J., and Nebert, D. W. (1985) Nucleic Acids Res. 13, 4503–4520. 8. Pfeffer, U., Fecarotta, E., and Vidali, G. (1995) BioTechniques 18, 204–206. 9. Jaiswal, A. K., Gonzalez, F. J., and Nebert, D. W. (1985) Science 228, 80–83. 10. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156– 159. 11. McDonnell, W. M., Scheiman, J. M., and Traber, P. G. (1992) Gastroenterology 103, 1509–1516. 12. Schweikl, H., Taylor, J. A., Kitareewan, S., Linko, P., Nagorney, D., and Goldstein, J. A. (1993) Pharmacogenetics 3, 239–249. 13. Wilhelmsson, A., Whitelaw, M. L., Gustafsson, J. A., and Poellinger, L. (1994) J. Biol. Chem. 269, 19028–19033.
abnt
AP: Anal Bio
NOTES & TIPS
IgG, alkaline phosphatase-conjugated BCIP/NBT as substrate (Sigma Chemical Co.)). In Fig. 1A the diffusion is very limited in comparison with the same reaction carried out directly on the plate (Fig. 1C). One of the advantages offered by this technique is the exact identification of the wild-type plaques which are to be avoided in further amplification. By this technique it is in fact possible to maintain under control the tendency of some clones to show occasionally the presence of b-galactosidase-positive plaques, a finding which may be the result of contamination with wild-type strains of the phage (presumably favored by a higher growth rate) or, less probable, by reversal to the wild type as a consequence of a precise excision at the cloning site of the cDNA insert. Perhaps the greatest advantage offered by the proposed technique concerns the possibility of running the control experiment in parallel with the traditional screening of the library in a very inexpensive way, in spare time without any need for extra cultures. We feel that it is always safer to have the experiment and the control carried out on the very same plate. REFERENCE 1. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Polymerase Chain Reaction (PCR)-Based Construction of a Competitor Target for Quantitative Reverse Transcriptase PCR Measurement of Cytochrome P450 1A1 mRNA Roland Fro¨tschl,* Ullrich Kleeberg,† Alfred G. Hildebrandt,† Ivar Roots,* and Ju¨rgen Brockmo¨ller* *Institute of Clinical Pharmacology, University Hospital Charite´, Humboldt University of Berlin, Schumannstrasse 20/21, D-10098 Berlin, Germany; and †Federal Institute of Drugs and Medical Products, Seestrasse 10, D-13353 Berlin, Germany Received March 26, 1996
Reliable quantification of mRNA by RT-PCR requires an internal or external standard cotranscribed and coamplified with the mRNA of interest. As internal standards provide only a relative quantification, external standards optimized for the assay of interest are required (1). Quantitative RT-PCR based on quantification of cDNA molecules transcribed from mRNA by using external DNA standards was already described ANALYTICAL BIOCHEMISTRY ARTICLE NO.
FIG. 1. PCR-mediated construction of artificial competitor mRNA. Steps of construction indicated as 1, 2, 3, and 4 and primers as well as gene and vector names and nucleotide positions are as described in the text.
by Gilliland et al. (2). However, different RT reaction efficiencies could not be excluded. Cytochrome P450 enzymes are important metabolizers of drugs, steroids, and xenobiotics, and as such they are of great medical interest (3). CYP1A1 is an important activator of polycyclic aromatic hydrocarbons and environmental pollutants (4), transforming such compounds into ultimate carcinogens (5). Therefore, it is of great clinical importance to evaluate the inducing potency of drugs and xenobiotics with high accuracy. Omeprazole is a known inducer of CYP 1A1 and 1A2 (6), but generally accepted systems to compare the inducing potency of omeprazole and related drugs (like the benzimidazolone domperidone) are still missing. Competitor artificial RNA (aRNA)1 was constructed from genomic CYP1A1, spanning the intron 6 from nucleotides 6435 to 6870 (7). After PCR of nucleotides 6435–6808, we distinguished between amplified contaminating genomic DNA and aRNA by an artificial EcoRI site introduced into the aRNA. The competitor aRNA was made in four steps (Fig. 1). Left and right arms were synthesized in two PCR (step 1), one with KON1(5*-ATCCTCTAGACCACCCGTTGCAGCAGGATAGCC-3*)/MU1 (5*-CCTACCTGAATTCTTTCTCACCCC-3*) and one with KON2 (5*-AAGTTCTGCAGGCTTTTACATCCCCAAGGGGCG-3*)/MU2 (5*-GGGGTGAGAAAGAATTCAGGTAGG-3*),each containing 1 mg of genomic DNA, 400 nM each primer, 80 mM each dNTP, 1.8 mM MgCl2 , 50 mM KCl, 10 mM Tris, pH 8.3, and 1 U of AmpliTaq DNA polymerase (Perkin–Elmer, Foster City, CA) in a final volume of 25 ml. Initial denaturation was 2 min at 957C, followed by 30 cycles with 1 min at 957C, 30 s at 527C, and 1 min at 727C and a final extension step at 727C for 7 min. PCR products were digested with EcoRI (Eurogen1
Abbreviations used: aRNA, artificial RNA; DMSO, dimethyl sulfoxide.
242, 280–282 (1996)
0468
0003-2697/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
10-18-96 11:54:42
abnt
AP: Anal Bio
281
NOTES & TIPS TABLE 1
Comparison of CYP1A1 mRNA Amounts in Hela Cells after Induction Inducer substance FIG. 2. RT-PCR of total RNA from Hela cells. One hundred nanograms of total RNA, DNAse digested, of omeprazole-induced (lanes 1 to 6) and domperidone-induced Hela cells (lanes 7 to 12). Serial dilutions of aRNA were added to the RT reactions: lanes 1 and 7, 40 fg; lanes 2 and 8, 8 fg; lanes 3 and 9, 1.6 fg; lanes 4 and 10, 0.32 fg; lanes 5 and 11, 0.064 fg; lanes 6 and 12, 0,0128 fg; lane M, 100-bp ladder.
Omeprazole Domperidone DMSO control
CYP1A1 mRNA CYP1A1 mRNA CYP1A1 mRNA per 100 ng molecules/100 ng molecules per total RNA total RNA cell 1.9 0.6 0.085
4600 1500
0.37 0.12
220
0.018
FIG. 3. Quantification of mRNA. Densitometer values of CYP1A1 mRNA-derived bands were plotted versus aRNA derived values. Curves result from linear regression.
gested with RNAse-free RQ1-DNAse (Promega) and quality was checked by gel electrophoresis. After purification on Ultrafree MC 30000 NMW filters, the amount of transcripts was quantitated by uv absorption at 260 nm. RT-reactions were set up according to the cDNA synthesis system manual of Life Technologies (Gaithersburg, MD), using MMLV-RT and a temperature-sensitive primer RT1 (5*-CAGGAAGAGAAAGAC-3*) (8). Reactions were adjusted to a final volume of 20 ml containing 8 U RNAsin (Promega) and 100 ng of total cellular RNA. Assays were overlaid with 25 ml mineral oil (Sigma, St. Louis, MO) and incubated at 377C for 1 h. Tubes were heated to 957C for 10 min and 30 ml of PCRmix was added at 957C, containing 50 mM KCl, 10 mM Tris, pH 8.3, 10 pmol of primers 1A8 (5*-GGCTTTTACATCCCCAAGGGGCG-3*) and 1A9 (5*-ATACACTTCCGCTTGCCCATGCC-3*), 0.5 mM MgCl2 , 67 mM each dNTP, and 1.25 U AmpliTaq. Cycling conditions were 1 min at 957C, 40 s at 667C, and 1 min 30 s at 727C for 30 cycles followed by 7 min at 727C. After EcoRI digestion the PCR products were separated on a 2% agarose gel. mRNA-derived amplificates show a 182-bp band, genomic DNA-derived amplificates a 373-bp band (7, 9), and artificial RNA-derived amplificates two bands of 288 and 85 bp. Equality of the 373- and 182-bp fragments in PCR amplification was ensured by amplifying both in one PCR and taking samples at different cycle numbers. Quotients of band intensities were the same at all cycles. Tests for intercalation of ethidium bromide showed that the same amounts of DNA of the different bands resulted in nearly equal densitometer values (data not shown). Serial dilutions of aRNA were added to a series of probes with constant RNA concentration and the resulting bands were recorded with a digital image system. Bands were integrated with the Quantiscan densitometry software from Biosoft, Cambridge, UK, and density values of 182-bp bands were corrected for size by addition of log a (a Å 288/182 Å 1.58) before correlation with 288-bp values. Concentration of mRNA was determined by linear regression analyses of aRNA intensities versus mRNA intensities.
AID
abnt
tec S.A., Seraing, Belgium) and purified. Left and right arms were ligated and the ligated fragment was amplified with primers KON1 and KON2 (step 2) as described above with an annealing temperature of 607C for 20 s. Five microliters of the PCR was digested with XbaI/PstI (Eurogentec), and the digested construct (now named 1A1Eco) was purified on Ultrafree MC 30000 NMW centrifugation filters (Millipore) and ligated into XbaI/PstI digested pT7T3 19U vector (Pharmacia, Uppsala, Sweden). The transcription target was amplified with primers pA1 (5*-GATTAAGTTGGGTAACGCCAGGG-3*) and pA2 (5*-TATGTTGTGTGGAATTGTGAGCG-3*) (step 3) as described above with an annealing step of 20 s at 607C. The amplificate was purified by agarose gel electrophoresis and filtration on Ultrafree MC units. The in vitro transcription (step 4) was carried out in a T7 in vitro transcription system from Promega (Madison, WI). Transcripts were di-
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10-18-96 11:54:42
AP: Anal Bio
282
NOTES & TIPS
Hela cells were cultured in DMEM, 10% (v/v) new born calf serum to near confluency and presumed inducers were added as 5 mM solutions in DMSO up to 50 mM final concentration of inducer. Controls were pretreated with the same amount of DMSO alone. Cells were incubated for 18 h at 377C in a 5% CO2 atmosphere and harvested by conventional trypsination, washed twice with Dulbecco’s PBS solution, and counted. Total RNA was isolated from cell pellets of 107 cells according to Chomczynski and Sacchi (10), digested with 10 U RNAse-free RQ1 DNAse (Promega) for 15 min at 377C, phenol–chloroform extracted, and ethanol precipitated. RNA concentration was determined spectrophotometrically and measured for 1A1 mRNA. Omitting the DNAse digestion resulted in contamination with genomic DNA, showing a 373-bp band in the RT-PCR assay. Results from induction tests of Hela cells with omeprazole and domperidone are demonstrated in Fig. 2. Fluorescence intensities of the CYP1A1 bands versus standard RNA bands were shown in Fig. 3. The number of standard RNA molecules of the intersection values of standard and mRNA curves was taken as the equilibrium value between mRNA molecules and standard molecules. Using serial dilutions with more narrow dilution steps (Fig. 3A) results in the same intersection values, demonstrating the high reliability and reproducibility of the measuring method. The described method is much more sensitive and absolutely selective compared to analysis by Northern blot or quantification of proteins with Western blot or enzyme-specific reporter reactions as already suggested by others (11, 12). Results of comparison of the intersection values in Fig. 3 are summarized in Table 1. Compared to uninduced controls we obtained a 22-fold increase in transcribed CYP1A1 mRNA for induction with omeprazole and a 7-fold induction with domperidone. The amount of 1A1 mRNA in omeprazole-induced Hela cells is about 4600 molecules or 1.9 fg per 100 ng of RNA. For approximately 2 to 5 ng of poly(A)/ mRNA out of 100 ng total cellular RNA, 1A1 mRNA is about 0.0001 to 0.00005%
AID
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/
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10-18-96 11:54:42
total mRNA. In relation to the cell number used for RNA purification, the 1A1 mRNA level in uninduced Hela cells is about 1 molecule per 57 cells and increases in omeprazole-induced cells to 1 molecule per 2.7 cells and in domperidone-induced cells to 1 molecule per 8.3 cells. The increase in amount of CYP1A1 mRNA in Hela cells upon exposure to omeprazole or domperidone may be due to AH receptor-mediated transcriptional activation (13). The enzyme-inducing activity of domperidone is new and illustrates a possible application of our mRNA amplification method for drug development. The threefold lower induction may be due to lower affinity of benzimidazolones to the AH receptor compared to benzimidazoles and the differentiation of compounds according to their potency in enzyme induction may be of great medical relevance. Expression levels of CYP1A1 are considered to modulate the risk of cancer. REFERENCES 1. Chelly, J., and Kahn, A. (1994) in The Polymerase Chain Reaction (Mullis, K. B., Ferre´, F., and Gibbs, R. A., Eds.), pp. 97– 109, Birkhauser, Boston, MA. 2. Gilliland, G., Perrin, S., Blanchard, K., and Bunn, H. F. (1990) Proc. Natl. Acad. Sci. USA 87, 2725–2729. 3. Estabrook, R. W. (1990) Biochem. Soc. Trans. 18, 34–36. 4. Catteau, A., Douriez, E., Beaune, P., Poisson, N., Bonaiti-Pellie´, C., and Laurent, P. (1995) Pharmacogenetics 5, 110–119. 5. Beresford, A. P. (1993) Drug Metab. Rev. 25, 503–517. 6. Rost, K. L., Bro¨sicke, H., Brockmo¨ller, J., Scheffler, M., Helge, H., and Roots, I. (1992) Clin. Pharmacol. Ther. 52, 170–180. 7. Jaiswal, A. K., Gonzalez, F. J., and Nebert, D. W. (1985) Nucleic Acids Res. 13, 4503–4520. 8. Pfeffer, U., Fecarotta, E., and Vidali, G. (1995) BioTechniques 18, 204–206. 9. Jaiswal, A. K., Gonzalez, F. J., and Nebert, D. W. (1985) Science 228, 80–83. 10. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156– 159. 11. McDonnell, W. M., Scheiman, J. M., and Traber, P. G. (1992) Gastroenterology 103, 1509–1516. 12. Schweikl, H., Taylor, J. A., Kitareewan, S., Linko, P., Nagorney, D., and Goldstein, J. A. (1993) Pharmacogenetics 3, 239–249. 13. Wilhelmsson, A., Whitelaw, M. L., Gustafsson, J. A., and Poellinger, L. (1994) J. Biol. Chem. 269, 19028–19033.
abnt
AP: Anal Bio