Nucleic Acids Research, Vol. 19, No. 13 3751
A method for the analysis of newly synthesized tritiated mRNA Torik A.Y.Ayoubi, Dominique P.V.de Kleijn, Eric W.Roubos and Gerard J.M.Martens Department of Animal Physiology, University of Nijmegen, Toernooiveld, 6525 ED Nijmegen, The Netherlands Submitted January 25, 1991 Molecular hybridization with RNA probes has been performed on unfractionated cells solubilized in guanidine thiocyanate solutions for the quantitative analysis of specific RNA transcripts (1, 2). A method utilizing this principle has been developed for the detection and quantification of newly synthesized mRNA. To evaluate the method, we analyzed the biosynthesis of proopiomelanocortin (POMC) mRNA in the neurointermediate lobe (NIL) of the pituitary gland of Xenopus laevis. Specificity of hybridization was assessed by comparing the results of sense and anti-sense probes at the experimentally determined optimal hybridization temperature of 35°C. Only the anti-sense probe protected newly synthesized [3H]-labeled POMC mRNA (Figure lA). Treatment of NILs during the labeling period with 5 ,6-dichloro- 1-f-D-ribofuranosylbenzimidazole (DRB), an mRNA synthesis inhibitor (3), decreased POMC mRNA synthesis by more than 90% (Figure IB). To show that the method is quantitative, increasing amounts of a [32P]-labeled Xenopus POMC cDNA fragment were assayed instead of labeled tissue. This resulted in a linear increase of hybridization signals (r = 0.9924, n = 3) (Figure 2). A single NIL was sufficient for detection. This tissue contains only ca. 6 x I04 melanotrope cells expressing the POMC gene. Therefore, the method seems to be particularly useful for studying small numbers of cells. The use of denaturing gels would enable mRNA and pre-mRNA to be distinguished. Since in guanidine thiocyanate solutions RNA -RNA hybrids are more stable than DNA -RNA hybrids (1), the use of cRNA probes might even
improve sensitivity.
ACKNOWLEDGEMENTS This study was supported by the Netherlands Organization for Scientific Research (NWO) and the European Community (Contract ST25-0468-C).
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100. Bo.
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10 15 20 slice number
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Figure 1. A. Specificity of hybridization signal. [3H]-labeled POMC mRNA was hybridized to the sense probe (ssM13DNA containing a 1 kb PstI-BglII POMC mRNA sequence) (4) or to the anti-sense probe (ssM13DNA containing complementary sequence). Results are shown for single NILs labeled for 8 h with [3H]uridine. B. Effect of 100 1M DRB on synthesis of POMC mRNA. Data represent the mean of 6 individual NILs ± SEM. NILs were labeled by incubation in Xenopus culture medium (5) with 1 mCi/ml [3H]uridine (46 Ci/mmol, Amersham Corp.) for 8 h at 22°C. Next, they were homogenized in 20 itl hybridization buffer (4 M guanidine thiocyanate; 25 mM sodium citrate pH 7.0; 0.5% Sarcosyl; 0.1 M 2-mercaptoethanol). After adding 1 tcg of sense or anti-sense probe, the homogenate was heated to 65°C for 10 min and hybridized for 16 h at 35°C. After hybridization, 0.7 ml 0.5 M NaCI, 20 Ag RNAse A and 20 U RNAse TI were added and the homogenate was incubated for 30 min at 37°C. Homogenates were phenol/chloroform extracted and isopropanol precipitated. The protected hybrids were analyzed on a 1 % agarose gel; gel slices were boiled for 15 min in 1 ml H20, and counted with a liquid scintillation analyser.
1 2 3 4 5 6
1234560
REFERENCES 1. Thompson,J. and Gillespie,D. (1987) Anal. Biochem. 163, 281-291. 2. Firestein,G.S., Gardner,S.M. and Roeder,W.D. (1987) Anal. Biochem. 167, 381 -386. 3. Zandomeni,R., Bunick,D., Ackerman,S., Mittleman,B. and Weinmann,R. (1983) J. Mol. Biol. 167, 561-574. 4. Martens,G.J.M. (1986) Nucl. Acids Res. 14, 3791-3798. 5. Ayoubi,T.A.Y., van Duijnhoven,H.L.P., van de Ven,W.J.M., Jenks,B.G., Roubos,E.W. and Martens,G.J.M. (1990) J. Biol. Chem. 265, 15644-15647.
Figure 2. Demonstration of the quantitative nature of the analysis. Each lane contains 1 lsg of the anti-sense M13 ssDNA, hybridized to increasing amounts of a PstI-EcoRI fragment of the Xenopus POMC cDNA which was labeled only on the sense strand by filling in of the EcoRI-site with DNA polymerase (Klenow fragment) and [32P]dATP. Increasing amounts of the labeled fragment were added to the hybridization buffer and assayed as described above. Lanes 1-6, 8000, 16000, 24000, 32000, 40000, 48000 cpm added, respectively.
A method for the analysis of newly synthesized tritiated mRNA Torik A.Y.Ayoubi, Dominique P.V.de Kleijn, Eric W.Roubos and Gerard J.M.Martens Department of Animal Physiology, University of Nijmegen, Toernooiveld, 6525 ED Nijmegen, The Netherlands Submitted January 25, 1991 Molecular hybridization with RNA probes has been performed on unfractionated cells solubilized in guanidine thiocyanate solutions for the quantitative analysis of specific RNA transcripts (1, 2). A method utilizing this principle has been developed for the detection and quantification of newly synthesized mRNA. To evaluate the method, we analyzed the biosynthesis of proopiomelanocortin (POMC) mRNA in the neurointermediate lobe (NIL) of the pituitary gland of Xenopus laevis. Specificity of hybridization was assessed by comparing the results of sense and anti-sense probes at the experimentally determined optimal hybridization temperature of 35°C. Only the anti-sense probe protected newly synthesized [3H]-labeled POMC mRNA (Figure lA). Treatment of NILs during the labeling period with 5 ,6-dichloro- 1-f-D-ribofuranosylbenzimidazole (DRB), an mRNA synthesis inhibitor (3), decreased POMC mRNA synthesis by more than 90% (Figure IB). To show that the method is quantitative, increasing amounts of a [32P]-labeled Xenopus POMC cDNA fragment were assayed instead of labeled tissue. This resulted in a linear increase of hybridization signals (r = 0.9924, n = 3) (Figure 2). A single NIL was sufficient for detection. This tissue contains only ca. 6 x I04 melanotrope cells expressing the POMC gene. Therefore, the method seems to be particularly useful for studying small numbers of cells. The use of denaturing gels would enable mRNA and pre-mRNA to be distinguished. Since in guanidine thiocyanate solutions RNA -RNA hybrids are more stable than DNA -RNA hybrids (1), the use of cRNA probes might even
improve sensitivity.
ACKNOWLEDGEMENTS This study was supported by the Netherlands Organization for Scientific Research (NWO) and the European Community (Contract ST25-0468-C).
.1400 1400
anti-sense
;"
< 1200
a
o
* sense
oool
Z
50O
100. Bo.
.800
60.8
.400
240-
20020 5
10 15 20 slice number
25
control ORB
Figure 1. A. Specificity of hybridization signal. [3H]-labeled POMC mRNA was hybridized to the sense probe (ssM13DNA containing a 1 kb PstI-BglII POMC mRNA sequence) (4) or to the anti-sense probe (ssM13DNA containing complementary sequence). Results are shown for single NILs labeled for 8 h with [3H]uridine. B. Effect of 100 1M DRB on synthesis of POMC mRNA. Data represent the mean of 6 individual NILs ± SEM. NILs were labeled by incubation in Xenopus culture medium (5) with 1 mCi/ml [3H]uridine (46 Ci/mmol, Amersham Corp.) for 8 h at 22°C. Next, they were homogenized in 20 itl hybridization buffer (4 M guanidine thiocyanate; 25 mM sodium citrate pH 7.0; 0.5% Sarcosyl; 0.1 M 2-mercaptoethanol). After adding 1 tcg of sense or anti-sense probe, the homogenate was heated to 65°C for 10 min and hybridized for 16 h at 35°C. After hybridization, 0.7 ml 0.5 M NaCI, 20 Ag RNAse A and 20 U RNAse TI were added and the homogenate was incubated for 30 min at 37°C. Homogenates were phenol/chloroform extracted and isopropanol precipitated. The protected hybrids were analyzed on a 1 % agarose gel; gel slices were boiled for 15 min in 1 ml H20, and counted with a liquid scintillation analyser.
1 2 3 4 5 6
1234560
REFERENCES 1. Thompson,J. and Gillespie,D. (1987) Anal. Biochem. 163, 281-291. 2. Firestein,G.S., Gardner,S.M. and Roeder,W.D. (1987) Anal. Biochem. 167, 381 -386. 3. Zandomeni,R., Bunick,D., Ackerman,S., Mittleman,B. and Weinmann,R. (1983) J. Mol. Biol. 167, 561-574. 4. Martens,G.J.M. (1986) Nucl. Acids Res. 14, 3791-3798. 5. Ayoubi,T.A.Y., van Duijnhoven,H.L.P., van de Ven,W.J.M., Jenks,B.G., Roubos,E.W. and Martens,G.J.M. (1990) J. Biol. Chem. 265, 15644-15647.
Figure 2. Demonstration of the quantitative nature of the analysis. Each lane contains 1 lsg of the anti-sense M13 ssDNA, hybridized to increasing amounts of a PstI-EcoRI fragment of the Xenopus POMC cDNA which was labeled only on the sense strand by filling in of the EcoRI-site with DNA polymerase (Klenow fragment) and [32P]dATP. Increasing amounts of the labeled fragment were added to the hybridization buffer and assayed as described above. Lanes 1-6, 8000, 16000, 24000, 32000, 40000, 48000 cpm added, respectively.
