Electrophoresis
1992, 13, 637-638
Nonradioactive ribonuclease protection assay
Nonradioactive ribonuclease protection analysis using digoxygenine labeling and chemiluminescent detection
Iris Wundrack Steven Dooley Institute of Human Genetics, Saar University, Homburg/Saar
A sensitive nonradioactive ribunouclease protection assay is described which we have used to study c-myc gene transcription and promoter usage in GLC4, a human small cell lung carcinoma cell line with amplified gene. For in vitro transcription we used digoxygenine (DIG)-rUTP instead of [cx-~~PICTP or [cx-~~PIUTP and after polyacrylamide gel electrophoresis the protected probes were transferred to a nylon membrane from Boehringer Mannheim using electroblotting. Subsequently the membrane was analyzed by chemiluminescent detection. Results were obtained after 1 h of exposure and were comparable with those using radioactivity.
The ribonuclease protection assay (RPA) is an extremely sensitive procedure for the detection and quantitation of mRNA species in a complex sample mixture of total cellular RNA. Compared to hybridization protocols that rely on RNA bound to a solid support.(i.e. Northern blots) low abundance mRNA’s are detected more readily and are quantitated more accurately by using a solution hybridization procedure such as RPA [l].Recombinants used in the RPA are prepared by cloning the sequence of interest downstream of a bacteriophage promoter oriented to generate an antisense probe of high specific activity. The plasmid is cleaved with a restriction enzyme, and incubated with bacteriophage RNA polymerase, which eficiently transcribes the cloned sequence into a discrete antisense RNA species. It is preferable to purify the probe before hybridization to sample RNAs. The hybridization reaction is treated with ribonuclease to remove free probe, leaving intact fragments of antisense RNA annealed to homologues sequences in the sample RNA. After purification these protected fragments were separated in a standard sequencing gel and visualized by autoradioagraphy. In another approach, by adjusting the ribonuclease concentration in the digestion reaction, the assay can be used to detect small differences between the probe and target mRNA. In this way, the RPA can also be used to map mutation sites [2-41. The disadvantage of the standard procedure is the handling with high specific 32P-labeledprobe. We therefore describe a sensitive nonradioactive ribonuclease protection assay that we have used to study c-myc gene transcription and promoter usage in GLC4, a human small cell lung carcinoma cell line with an amplified gene (Fig. 1). The new procedure described here as well as the radioactive control experiment were performed with the components of an RNase Protection Kit (Ambion) and generally can be divided into three stages: (i) Probe preparation: template DNA is prepared by inserting an MboI-T’qI fragment of the c-myc gene’s regulatory region into the plasmid vector pSPT19 under the control of
Correspondence: Dr. Steven Dooley, Institute of Human Genetics, Saar University, DW-6650 Homburg/Saar, Germany Abbreviations: DIG, dioxygenine; RPA, ribonuclease protection analysis
0VCH Verlagsgesellschaft
637
mbH, D-6940 Weinheirn, 1992
T7 bacteriophage promoter [ 5 ] .The digestion of the DNA with the restriction enzyme EcoRI that cuts immediately downstream of the insert sequence, and the subsequent in vitro transcription with T7 RNA polymerase, will allow the generation of a uniquely sized runoff antisense transcript. We used 10 pg of template DNA in a 10 pL transcription reaction containing 2 mM rATP, rGTP, rCTP, 0.7 mM digoxygenine (DIG)-rUTP and 1.3 mM unlabeled rUTP; the DIGrUTP:rUTP ratio should be 0.35 : 0.65. The T7 RNA polymerase generates an antisense RNA transcript, of which 302 nucleotides will be protected by c-myc mRNA target. After incubation for 1 h at 37”C,probes were treated with 1 unit RNase-free DNase I(15 min at 37°C) to destroy DNA template. To remove virtually free nucleoside triphosphates from the polymerized RNAwe recommend that the probe should be purified by column purification (“Push Column”, Stratagene). (ii) Solution hybridization and RNase digestion: the amount of labeled RNA transcript generated can be approximated by comparison with DIG-labeled control-DNA in a dot blot (DIG Nucleic Acid Detection Kit, Boehringer Mannheim, BM). Hybridization was performed by suspending 10 pg sample RNA in 20 pL hybridization buffer (80 O/o formamide, 40 mM piperazine-hi,”-bis (2-ethanesulfonic acid) (PIPES), pH 6.4,400 mM NaOAc, pH 6.4, 1 mM EDTA), adding about 500 pg DIG-labeled RNA probe and denaturing the mixture for5 rnin at 90°C. The samples were immediately transferred to 42°C and incubated overnight. After annealing, 200 pL RNase digestion buffer (solution Bx,Ambion kit) containing RNase A(0.5 U/mLand RNase T I (100 U/mL) was added to the hybridization mixture. Samples were mixed by briefly vortexing and incubated for 30 min at 37°C. The RNase reaction was terminated by addition of 300 pL RNase inactivation/precipitation mixture (solution Dx, Ambion kit) and subsequently transferred to -20°C freezer for at least 15 min. The tubes were removed from the freezer and were microfuged for 15 min at maximum speed and 4°C. Supernatant is carefully and completely removed from each tube because residual supernatant results in aberrant migration of bands in the gel. (iii) Polyacrylamide gel electrophoresis, electroblotting and detection: the dried RNA pellet is resuspended in 5 pL RNA loading buffer (80% formamide, 0.1 Yo Xylene Cyanol, 0.1% Bromphenol Blue, 2 mM EDTA) and incubated in 95”C for 10 min. Undissolved material is pelleted by microfuge centrifugation (5 min) and supernatant is transferred to a new tube. Subsequently the samples are denatured 0173-0835/92/0910-0637 $3.50+.25/0
638
1. Wundrack and S. Dooley
1
2
EIectrophoreJis 1992, 13, 631-638
3
4
5
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30213--
again for 5 min at 95" C, put on ice and directly loaded on a preelectrophoresed 6% sequencing gel. The gel is run at 250 Vin 1 X TBE (Tris-borate-EDTA) for about 1 h in a Protean I1 electrophoresis apparatus (Bio-Rad), using 0.75 m m spacers. After electrophoresis the gel is soaked for 10 min in 0.5 X TBE and blotted onto BM nylon membrane with the Trans-Blot S D Semi-Dry Electrophoretic Transfer Cell (Bio-Rad), according to the manufacturer's instructions in the "Trans-Blot S D DNABlotting Kit".The radioactive control part ofthe gel is fixed in 10% methanol/l0% acetic acid and dried in a gel dryer for 2 h at 80 "C. After electrotransfer the bands on the filter are detected with the DIG nucleic acid detection kit, according to the chemiluminescent detection protocol. Autoradiography was performed for 1-2 h to measure chemiluminescence, and overnight for radioactive probes. Data indicate thal, using DIG-rUTP labeled riboprobes and chemiluminescent detection for RPA analysis, results were comparable to those with radioactively labeled ones.
M
--309b
Figure 1. RNase protection analysis using pM 302/T7 transcripts, corresponding io the 302 bp c-myc promotor region and different RNA probes. M. Marker pBR322/ HpaII labeled with (32p-)dCTPand Klenow fragmeni; (1) c-myc overexpressing small cell lung cancer cell line GLC4 RNA, untreated cells; (2) quiescent cells; (3) serum cells stimulated (20 niin); (4) serum cells stimulated (90 min). Samples (1)-(4) were analyzed by using a DIG-labeled probe and were detected with chemiluminescence. (5)-(8) correspond to (1)-(4), using radioactive label; 302b indicates the size of the protected fragment, 309b is the marker band.
Clear signals are detectable with high signal-to-noise ratio. Nonradioactive DIG-labeled probes can be stored and used indefinitely without having to worry about sensitivity loss and sample degradation. A further advantage is the short exposure time of 1-2 h. In our view the only disadvantage is the fact that the DIG-labeled probe cannot be purified before hybridization, occasionally resulting in background problems.
References [ l ] Lee, J.J. and Costlow, N.A., Methods Enzy'mol. 1987. 152, 633-648. [2] Myers, R.M. and Maniatis, T., Cold Spring HarborSymposia o/Quantitative Biology, 1986 51, 275-284. [ 3 ] Winter, E.,Yamamoto,F.,Almoguera,C. and Perucho,M.,Proc/. Nut/. Acad. Sci. USA 1985, 82,7575-7580. [4] Genovese, C., Brufsky, A,, Shapiro, .I.and Rowe, D., J. Biol.Chem. 1989,264,9632-9637. [5] Polack, A . , Strobl, L., Feederle, R., Schweizer, M., Koch, E.,Eick, D., Wiegand, H. and Bornkamm, G.W., Oncogene 1991, 6,2033-2040.
1992, 13, 637-638
Nonradioactive ribonuclease protection assay
Nonradioactive ribonuclease protection analysis using digoxygenine labeling and chemiluminescent detection
Iris Wundrack Steven Dooley Institute of Human Genetics, Saar University, Homburg/Saar
A sensitive nonradioactive ribunouclease protection assay is described which we have used to study c-myc gene transcription and promoter usage in GLC4, a human small cell lung carcinoma cell line with amplified gene. For in vitro transcription we used digoxygenine (DIG)-rUTP instead of [cx-~~PICTP or [cx-~~PIUTP and after polyacrylamide gel electrophoresis the protected probes were transferred to a nylon membrane from Boehringer Mannheim using electroblotting. Subsequently the membrane was analyzed by chemiluminescent detection. Results were obtained after 1 h of exposure and were comparable with those using radioactivity.
The ribonuclease protection assay (RPA) is an extremely sensitive procedure for the detection and quantitation of mRNA species in a complex sample mixture of total cellular RNA. Compared to hybridization protocols that rely on RNA bound to a solid support.(i.e. Northern blots) low abundance mRNA’s are detected more readily and are quantitated more accurately by using a solution hybridization procedure such as RPA [l].Recombinants used in the RPA are prepared by cloning the sequence of interest downstream of a bacteriophage promoter oriented to generate an antisense probe of high specific activity. The plasmid is cleaved with a restriction enzyme, and incubated with bacteriophage RNA polymerase, which eficiently transcribes the cloned sequence into a discrete antisense RNA species. It is preferable to purify the probe before hybridization to sample RNAs. The hybridization reaction is treated with ribonuclease to remove free probe, leaving intact fragments of antisense RNA annealed to homologues sequences in the sample RNA. After purification these protected fragments were separated in a standard sequencing gel and visualized by autoradioagraphy. In another approach, by adjusting the ribonuclease concentration in the digestion reaction, the assay can be used to detect small differences between the probe and target mRNA. In this way, the RPA can also be used to map mutation sites [2-41. The disadvantage of the standard procedure is the handling with high specific 32P-labeledprobe. We therefore describe a sensitive nonradioactive ribonuclease protection assay that we have used to study c-myc gene transcription and promoter usage in GLC4, a human small cell lung carcinoma cell line with an amplified gene (Fig. 1). The new procedure described here as well as the radioactive control experiment were performed with the components of an RNase Protection Kit (Ambion) and generally can be divided into three stages: (i) Probe preparation: template DNA is prepared by inserting an MboI-T’qI fragment of the c-myc gene’s regulatory region into the plasmid vector pSPT19 under the control of
Correspondence: Dr. Steven Dooley, Institute of Human Genetics, Saar University, DW-6650 Homburg/Saar, Germany Abbreviations: DIG, dioxygenine; RPA, ribonuclease protection analysis
0VCH Verlagsgesellschaft
637
mbH, D-6940 Weinheirn, 1992
T7 bacteriophage promoter [ 5 ] .The digestion of the DNA with the restriction enzyme EcoRI that cuts immediately downstream of the insert sequence, and the subsequent in vitro transcription with T7 RNA polymerase, will allow the generation of a uniquely sized runoff antisense transcript. We used 10 pg of template DNA in a 10 pL transcription reaction containing 2 mM rATP, rGTP, rCTP, 0.7 mM digoxygenine (DIG)-rUTP and 1.3 mM unlabeled rUTP; the DIGrUTP:rUTP ratio should be 0.35 : 0.65. The T7 RNA polymerase generates an antisense RNA transcript, of which 302 nucleotides will be protected by c-myc mRNA target. After incubation for 1 h at 37”C,probes were treated with 1 unit RNase-free DNase I(15 min at 37°C) to destroy DNA template. To remove virtually free nucleoside triphosphates from the polymerized RNAwe recommend that the probe should be purified by column purification (“Push Column”, Stratagene). (ii) Solution hybridization and RNase digestion: the amount of labeled RNA transcript generated can be approximated by comparison with DIG-labeled control-DNA in a dot blot (DIG Nucleic Acid Detection Kit, Boehringer Mannheim, BM). Hybridization was performed by suspending 10 pg sample RNA in 20 pL hybridization buffer (80 O/o formamide, 40 mM piperazine-hi,”-bis (2-ethanesulfonic acid) (PIPES), pH 6.4,400 mM NaOAc, pH 6.4, 1 mM EDTA), adding about 500 pg DIG-labeled RNA probe and denaturing the mixture for5 rnin at 90°C. The samples were immediately transferred to 42°C and incubated overnight. After annealing, 200 pL RNase digestion buffer (solution Bx,Ambion kit) containing RNase A(0.5 U/mLand RNase T I (100 U/mL) was added to the hybridization mixture. Samples were mixed by briefly vortexing and incubated for 30 min at 37°C. The RNase reaction was terminated by addition of 300 pL RNase inactivation/precipitation mixture (solution Dx, Ambion kit) and subsequently transferred to -20°C freezer for at least 15 min. The tubes were removed from the freezer and were microfuged for 15 min at maximum speed and 4°C. Supernatant is carefully and completely removed from each tube because residual supernatant results in aberrant migration of bands in the gel. (iii) Polyacrylamide gel electrophoresis, electroblotting and detection: the dried RNA pellet is resuspended in 5 pL RNA loading buffer (80% formamide, 0.1 Yo Xylene Cyanol, 0.1% Bromphenol Blue, 2 mM EDTA) and incubated in 95”C for 10 min. Undissolved material is pelleted by microfuge centrifugation (5 min) and supernatant is transferred to a new tube. Subsequently the samples are denatured 0173-0835/92/0910-0637 $3.50+.25/0
638
1. Wundrack and S. Dooley
1
2
EIectrophoreJis 1992, 13, 631-638
3
4
5
6
7
8
30213--
again for 5 min at 95" C, put on ice and directly loaded on a preelectrophoresed 6% sequencing gel. The gel is run at 250 Vin 1 X TBE (Tris-borate-EDTA) for about 1 h in a Protean I1 electrophoresis apparatus (Bio-Rad), using 0.75 m m spacers. After electrophoresis the gel is soaked for 10 min in 0.5 X TBE and blotted onto BM nylon membrane with the Trans-Blot S D Semi-Dry Electrophoretic Transfer Cell (Bio-Rad), according to the manufacturer's instructions in the "Trans-Blot S D DNABlotting Kit".The radioactive control part ofthe gel is fixed in 10% methanol/l0% acetic acid and dried in a gel dryer for 2 h at 80 "C. After electrotransfer the bands on the filter are detected with the DIG nucleic acid detection kit, according to the chemiluminescent detection protocol. Autoradiography was performed for 1-2 h to measure chemiluminescence, and overnight for radioactive probes. Data indicate thal, using DIG-rUTP labeled riboprobes and chemiluminescent detection for RPA analysis, results were comparable to those with radioactively labeled ones.
M
--309b
Figure 1. RNase protection analysis using pM 302/T7 transcripts, corresponding io the 302 bp c-myc promotor region and different RNA probes. M. Marker pBR322/ HpaII labeled with (32p-)dCTPand Klenow fragmeni; (1) c-myc overexpressing small cell lung cancer cell line GLC4 RNA, untreated cells; (2) quiescent cells; (3) serum cells stimulated (20 niin); (4) serum cells stimulated (90 min). Samples (1)-(4) were analyzed by using a DIG-labeled probe and were detected with chemiluminescence. (5)-(8) correspond to (1)-(4), using radioactive label; 302b indicates the size of the protected fragment, 309b is the marker band.
Clear signals are detectable with high signal-to-noise ratio. Nonradioactive DIG-labeled probes can be stored and used indefinitely without having to worry about sensitivity loss and sample degradation. A further advantage is the short exposure time of 1-2 h. In our view the only disadvantage is the fact that the DIG-labeled probe cannot be purified before hybridization, occasionally resulting in background problems.
References [ l ] Lee, J.J. and Costlow, N.A., Methods Enzy'mol. 1987. 152, 633-648. [2] Myers, R.M. and Maniatis, T., Cold Spring HarborSymposia o/Quantitative Biology, 1986 51, 275-284. [ 3 ] Winter, E.,Yamamoto,F.,Almoguera,C. and Perucho,M.,Proc/. Nut/. Acad. Sci. USA 1985, 82,7575-7580. [4] Genovese, C., Brufsky, A,, Shapiro, .I.and Rowe, D., J. Biol.Chem. 1989,264,9632-9637. [5] Polack, A . , Strobl, L., Feederle, R., Schweizer, M., Koch, E.,Eick, D., Wiegand, H. and Bornkamm, G.W., Oncogene 1991, 6,2033-2040.
