- Email: [email protected]
Isolation of Total RNA fromArabidopsis thalianaSeeds
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NOTES & TIPS
Isolation of Total RNA from Arabidopsis thaliana Seeds 1 Carlos M. Vicient 2 and Michel Delseny Laboratoire de Physiologie et Biologie Mole´culaire des Plantes, Centre national de la Recherche Scientifique URA 565, Universite´ de Perpignan, 66860 Perpignan Cedex, France Received October 26, 1998
Arabidopsis (Arabidopsis thaliana L. Heynh) is widely used as a model organism in plant molecular biology (1, 3). Molecular biology is often focused on gene expression and requires RNA isolation. Our laboratory has been studying the gene expression during seed desiccation in Arabidopsis (2) and the efficient extraction of RNA from dry seeds, a task previously proven to be difficult. Dry seeds have a high content of storage lipids, storage proteins, and secondary metabolites such as mucilages and phenolics (7) which make RNA purification difficult. Moreover, the small size of Arabidopsis seeds limits the quantity of starting material. The method we present here is a modification of a method reported by Witchel and co-workers (10) for the preparation of RNA from marine invertebrates. This method is based on the use of 8 M LiCl in the extraction buffer followed by phenol extractions. The differences with respect to the original method are the inclusion of 2% b-mercaptoethanol in the solubilization buffer to avoid RNA degradation, a reduction in the number of phenol extractions, the addition of quartz powder to make easier the grinding of the hard seed coats, and a reduction in the volumes used. The original method used as much as 500 ml of initial volume, whereas we have adapted it to microcentrifuge tubes. We used 100 mg of seeds as starting material. Seeds were ground to a fine powder using mortar and pestle in the presence of liquid nitrogen. Due to the presence of the seed coat, grinding may be difficult and we recommend the addition of a small quantity of sterile quartz powder. Frozen ground seeds were transferred to 2-ml microcentrifuge tubes containing 2 ml of extraction buffer (8 M LiCl, 2% b-mercaptoethanol) precooled to 220°C, and the powder was totally resus1
C.V. was recipient of a postdoctoral fellowship from the Spanish Instituto Nacional de Investigacio´n y Tecnologı´a Agraria y Alimentaria (INIA). This work was supported by the CNRS and by the EC BIOTECH Program (Grant BIO4-CT96-0062), and benefits from the joint CNRS-CSIC Laboratoire Europe´en Associe´ Perpignan-Barcelone for Plant Molecular and Cellular Biology. 2 To whom correspondence should be addressed at current address: Institute of Biotechnology, University of Helsinki, Biocenter 1, P.O. Box 56, Viikinkaari 9, FIN-00014 Helsinki, Finland. Fax 1358 9 708 59570. E-mail: [email protected]. Analytical Biochemistry 268, 412– 413 (1999) Article ID abio.1998.3045 0003-2697/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
pended. After overnight incubation at 4°C we centrifuged the mixture for about 4 s and transferred the supernatant to clean 2-ml microcentrifuge tubes. At this step it is difficult to avoid collecting some of the insoluble residues but this is not critical because the following phenol extractions will eliminate them. The first supernatant was then centrifuged at 13,000 rpm for 30 min at 4°C, the resulting supernatant was decanted, and the pellet was washed with cold (4°C) 70% ethanol and briefly air-dried. We dissolved the pellet in 1 ml of solubilization buffer (0.5% SDS, 100 mM NaCl, 25 mM EDTA, 10 mM Tris–HCl, pH 7.6, 2% b-mercaptoethanol). It was then extracted twice with an equal volume of phenol equilibrated at pH 7.6, once with phenol:chloroform:isoamyl alcohol (25:24:1), and twice with chloroform:isoamyl alcohol (24:1). We avoid putting the samples on ice during these extractions to prevent SDS precipitation. In each case, organic and aqueous phases were mixed by hand agitation and then spun at 13,000 rpm for 15 min at 4°C. The last aqueous phase was distributed between two 1.5-ml microcentrifuge tubes and 0.1 vol of 3 M sodium acetate and 1.5 vol of ethanol were then added. The samples could be stored at 220°C at this point. The tubes were then centrifuged at 13,000 rpm for 30 min at 4°C, the supernatant was poured off, and 0.5 ml of 3 M sodium acetate was added. The pellet was vortexed for 1 min and centrifuged at 13,000 rpm for 10 min at 4°C, then washed with 70% ethanol, and dissolved in 100 ml of water. We used 2 ml of the sample for spectrophotometric analysis and 1 to 5 ml for electrophoresis in 1.2% agarose gel in 0.53 TBE buffer in the presence of ethidium bromide. The method described here is based on the use of a high concentration of lithium chloride in the extraction buffer. It does not eliminate nucleases until a second step but the presence of b-mercaptoethanol and probably the high salt concentration used in the extraction buffer (8 M LiCl) inhibit nuclease activity (8). Starting with 100 mg of Arabidopsis seeds and using this method, the yield of total RNA ranged from 15 to 20 mg, whereas with other methods the yields were very much lower. For comparison, using the guanidine– hydrochloride method (6), we obtained less than 1 mg RNA starting with the same amount of seeds. To test the quality of the RNA, two assays were performed: nondenaturing gel electrophoresis and Northern blots with an AtEm6 gene probe. RNA prepared by this protocol was intact as observed by agarose electrophoresis (Fig. 1A). The lack of fluorescence in the wells should also be noted. This and a good correlation between the spectrophotometer measurement and ethidium bromide staining suggest a high degree of purity in the RNA. Other indications are the similar intensities of the bands corresponding to the 25S and 18S rRNAs and the lack of a smear. For Northern blot hybridization, 2
NOTES & TIPS
FIG. 1. RNA quality. (A) Total RNA extracted from dry seeds of Arabidopsis thaliana subjected to 1.2% agarose gel electrophoresis in TBE buffer stained with ethidium bromide. (B) Northern blot hybridization of total RNA (2 mg) with an Arabidopsis [a- 32P]dCTPlabeled AtEm6 probe.
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extraction from seeds of tobacco (Nicotiana tabacum L.), holm oak (Quercus ilex L.), and almond (Prunus amygdalus Batsch), and from leaves, stems, flowers, and pollen of rapeseed. This method also performed well with potato (Solanum tuberosum L.) seeds (E. Carrera, CID-CSIC, Barcelona, personal communication). In summary, we have developed an improved method for total RNA extraction from seeds of Arabidopsis thaliana and other species satisfying yield and quality criteria. Acknowledgments. Special thanks to Dr. T. J. Roscoe and Dr. Alan H. Schulman for critical reading of the manuscript.
REFERENCES
mg total RNA was fractionated on a 0.8% agarose–2.2 M formaldehyde gel and immobilized on nylon membrane (Amersham). The AtEm6 gene, used as a probe, is an Arabidopsis gene coding for an Em-like protein which is highly expressed during the late stages of seed development. High levels of its mRNA are accumulated in dry seed (5). The exact function of Em proteins is not known, but on the basis of their hydrophilic properties and pattern of accumulation they are supposed to provide desiccation tolerance to the embryo (4). Labeling, hybridization, and detection were performed as described (9). As shown in Fig. 1B, a hybridization band corresponding to the intact mRNA was obtained. We have also used this method successfully with many other Arabidopsis cDNA probes (Vicient et al., unpublished results). We also tested the quality of this extraction method in seeds of other species and other plant tissues. For example, this method was also tested successfully with rapeseed (Brassica napus L.) and the yield of total RNA obtained from 100 mg of seeds ranged from 18 to 32 mg. We used this method successfully for the RNA
1. Bowman, J. (1993) Arabidopsis: An Atlas of Morphology and Development, Springer-Verlag, Berlin. 2. Delseny, M., Gaubier, P., Hull, G., Saez-Vazquez, J., Gallois, P., Raynal, M., Cooke, R., and Grellet, F. (1994) in Stress-Induced Gene Expression in Plants (Basra, A. S., Ed.), pp. 25–59, Harwood Academic Publishers, Reading. 3. Delseny, M., Cooke, R., Raynal, M., and Grellet, F. (1997) FEBS Lett. 405, 129 –132. 4. Dure, L., III, Crouch, M., Harada, J., Ho, T. H. D., Mundy, J., Quatrano, R., Thomas, T., and Sung. Z. R. (1989) Plant Mol. Biol. 12, 475– 486. 5. Gaubier, P., Raynal, M., Hull, G., Huestis, G., Grellet, F., Arenas, C., Pages, M., and Delseny, M. (1993) Mol. Gen. Genet. 238, 409 – 418. 6. Logemann, J., Schell, J., and Willmitzer L. (1987) Ann. Biochem. 163, 16 –20. 7. Meyerowitz, E. M., and Somerville, C. R. (1994). Arabidopsis. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 8. Record, M. Y., Lohman, T. M., and de Haseth, P. (1976) J. Mol. Biol. 107, 145–158. 9. Sambrook, J., Fritsch, E. F., and Maniatis T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 10. Witchel, H. J., Maitland N. J., and Meech, R. W. (1996) BioTechniques 21, 1024 –1026.
NOTES & TIPS
Isolation of Total RNA from Arabidopsis thaliana Seeds 1 Carlos M. Vicient 2 and Michel Delseny Laboratoire de Physiologie et Biologie Mole´culaire des Plantes, Centre national de la Recherche Scientifique URA 565, Universite´ de Perpignan, 66860 Perpignan Cedex, France Received October 26, 1998
Arabidopsis (Arabidopsis thaliana L. Heynh) is widely used as a model organism in plant molecular biology (1, 3). Molecular biology is often focused on gene expression and requires RNA isolation. Our laboratory has been studying the gene expression during seed desiccation in Arabidopsis (2) and the efficient extraction of RNA from dry seeds, a task previously proven to be difficult. Dry seeds have a high content of storage lipids, storage proteins, and secondary metabolites such as mucilages and phenolics (7) which make RNA purification difficult. Moreover, the small size of Arabidopsis seeds limits the quantity of starting material. The method we present here is a modification of a method reported by Witchel and co-workers (10) for the preparation of RNA from marine invertebrates. This method is based on the use of 8 M LiCl in the extraction buffer followed by phenol extractions. The differences with respect to the original method are the inclusion of 2% b-mercaptoethanol in the solubilization buffer to avoid RNA degradation, a reduction in the number of phenol extractions, the addition of quartz powder to make easier the grinding of the hard seed coats, and a reduction in the volumes used. The original method used as much as 500 ml of initial volume, whereas we have adapted it to microcentrifuge tubes. We used 100 mg of seeds as starting material. Seeds were ground to a fine powder using mortar and pestle in the presence of liquid nitrogen. Due to the presence of the seed coat, grinding may be difficult and we recommend the addition of a small quantity of sterile quartz powder. Frozen ground seeds were transferred to 2-ml microcentrifuge tubes containing 2 ml of extraction buffer (8 M LiCl, 2% b-mercaptoethanol) precooled to 220°C, and the powder was totally resus1
C.V. was recipient of a postdoctoral fellowship from the Spanish Instituto Nacional de Investigacio´n y Tecnologı´a Agraria y Alimentaria (INIA). This work was supported by the CNRS and by the EC BIOTECH Program (Grant BIO4-CT96-0062), and benefits from the joint CNRS-CSIC Laboratoire Europe´en Associe´ Perpignan-Barcelone for Plant Molecular and Cellular Biology. 2 To whom correspondence should be addressed at current address: Institute of Biotechnology, University of Helsinki, Biocenter 1, P.O. Box 56, Viikinkaari 9, FIN-00014 Helsinki, Finland. Fax 1358 9 708 59570. E-mail: [email protected]. Analytical Biochemistry 268, 412– 413 (1999) Article ID abio.1998.3045 0003-2697/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
pended. After overnight incubation at 4°C we centrifuged the mixture for about 4 s and transferred the supernatant to clean 2-ml microcentrifuge tubes. At this step it is difficult to avoid collecting some of the insoluble residues but this is not critical because the following phenol extractions will eliminate them. The first supernatant was then centrifuged at 13,000 rpm for 30 min at 4°C, the resulting supernatant was decanted, and the pellet was washed with cold (4°C) 70% ethanol and briefly air-dried. We dissolved the pellet in 1 ml of solubilization buffer (0.5% SDS, 100 mM NaCl, 25 mM EDTA, 10 mM Tris–HCl, pH 7.6, 2% b-mercaptoethanol). It was then extracted twice with an equal volume of phenol equilibrated at pH 7.6, once with phenol:chloroform:isoamyl alcohol (25:24:1), and twice with chloroform:isoamyl alcohol (24:1). We avoid putting the samples on ice during these extractions to prevent SDS precipitation. In each case, organic and aqueous phases were mixed by hand agitation and then spun at 13,000 rpm for 15 min at 4°C. The last aqueous phase was distributed between two 1.5-ml microcentrifuge tubes and 0.1 vol of 3 M sodium acetate and 1.5 vol of ethanol were then added. The samples could be stored at 220°C at this point. The tubes were then centrifuged at 13,000 rpm for 30 min at 4°C, the supernatant was poured off, and 0.5 ml of 3 M sodium acetate was added. The pellet was vortexed for 1 min and centrifuged at 13,000 rpm for 10 min at 4°C, then washed with 70% ethanol, and dissolved in 100 ml of water. We used 2 ml of the sample for spectrophotometric analysis and 1 to 5 ml for electrophoresis in 1.2% agarose gel in 0.53 TBE buffer in the presence of ethidium bromide. The method described here is based on the use of a high concentration of lithium chloride in the extraction buffer. It does not eliminate nucleases until a second step but the presence of b-mercaptoethanol and probably the high salt concentration used in the extraction buffer (8 M LiCl) inhibit nuclease activity (8). Starting with 100 mg of Arabidopsis seeds and using this method, the yield of total RNA ranged from 15 to 20 mg, whereas with other methods the yields were very much lower. For comparison, using the guanidine– hydrochloride method (6), we obtained less than 1 mg RNA starting with the same amount of seeds. To test the quality of the RNA, two assays were performed: nondenaturing gel electrophoresis and Northern blots with an AtEm6 gene probe. RNA prepared by this protocol was intact as observed by agarose electrophoresis (Fig. 1A). The lack of fluorescence in the wells should also be noted. This and a good correlation between the spectrophotometer measurement and ethidium bromide staining suggest a high degree of purity in the RNA. Other indications are the similar intensities of the bands corresponding to the 25S and 18S rRNAs and the lack of a smear. For Northern blot hybridization, 2
NOTES & TIPS
FIG. 1. RNA quality. (A) Total RNA extracted from dry seeds of Arabidopsis thaliana subjected to 1.2% agarose gel electrophoresis in TBE buffer stained with ethidium bromide. (B) Northern blot hybridization of total RNA (2 mg) with an Arabidopsis [a- 32P]dCTPlabeled AtEm6 probe.
413
extraction from seeds of tobacco (Nicotiana tabacum L.), holm oak (Quercus ilex L.), and almond (Prunus amygdalus Batsch), and from leaves, stems, flowers, and pollen of rapeseed. This method also performed well with potato (Solanum tuberosum L.) seeds (E. Carrera, CID-CSIC, Barcelona, personal communication). In summary, we have developed an improved method for total RNA extraction from seeds of Arabidopsis thaliana and other species satisfying yield and quality criteria. Acknowledgments. Special thanks to Dr. T. J. Roscoe and Dr. Alan H. Schulman for critical reading of the manuscript.
REFERENCES
mg total RNA was fractionated on a 0.8% agarose–2.2 M formaldehyde gel and immobilized on nylon membrane (Amersham). The AtEm6 gene, used as a probe, is an Arabidopsis gene coding for an Em-like protein which is highly expressed during the late stages of seed development. High levels of its mRNA are accumulated in dry seed (5). The exact function of Em proteins is not known, but on the basis of their hydrophilic properties and pattern of accumulation they are supposed to provide desiccation tolerance to the embryo (4). Labeling, hybridization, and detection were performed as described (9). As shown in Fig. 1B, a hybridization band corresponding to the intact mRNA was obtained. We have also used this method successfully with many other Arabidopsis cDNA probes (Vicient et al., unpublished results). We also tested the quality of this extraction method in seeds of other species and other plant tissues. For example, this method was also tested successfully with rapeseed (Brassica napus L.) and the yield of total RNA obtained from 100 mg of seeds ranged from 18 to 32 mg. We used this method successfully for the RNA
1. Bowman, J. (1993) Arabidopsis: An Atlas of Morphology and Development, Springer-Verlag, Berlin. 2. Delseny, M., Gaubier, P., Hull, G., Saez-Vazquez, J., Gallois, P., Raynal, M., Cooke, R., and Grellet, F. (1994) in Stress-Induced Gene Expression in Plants (Basra, A. S., Ed.), pp. 25–59, Harwood Academic Publishers, Reading. 3. Delseny, M., Cooke, R., Raynal, M., and Grellet, F. (1997) FEBS Lett. 405, 129 –132. 4. Dure, L., III, Crouch, M., Harada, J., Ho, T. H. D., Mundy, J., Quatrano, R., Thomas, T., and Sung. Z. R. (1989) Plant Mol. Biol. 12, 475– 486. 5. Gaubier, P., Raynal, M., Hull, G., Huestis, G., Grellet, F., Arenas, C., Pages, M., and Delseny, M. (1993) Mol. Gen. Genet. 238, 409 – 418. 6. Logemann, J., Schell, J., and Willmitzer L. (1987) Ann. Biochem. 163, 16 –20. 7. Meyerowitz, E. M., and Somerville, C. R. (1994). Arabidopsis. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 8. Record, M. Y., Lohman, T. M., and de Haseth, P. (1976) J. Mol. Biol. 107, 145–158. 9. Sambrook, J., Fritsch, E. F., and Maniatis T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 10. Witchel, H. J., Maitland N. J., and Meech, R. W. (1996) BioTechniques 21, 1024 –1026.