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Simple purification of small RNAs from seeds and efficient detection of multiple microRNAs expressed in Arabidopsis thaliana and tomato (Lycopersicon esculentum) seeds

  • Ruth C. Martin (a1), Po-Pu Liu (a1) and Hiroyuki Nonogaki (a1)

Abstract

MicroRNAs (miRNAs) play critical roles in the development of animals and plants. Characterizing the stage- and tissue-specific expression of miRNAs that potentially regulate target transcription factor expression is becoming more important for understanding the regulatory mechanisms of critical events during plant development. A simple method for purifying small RNAs from seeds is described, as well as an efficient non-radioactive labelling system for making miRNA probes. In Arabidopsis thaliana seed extracts, low molecular-weight (LMW) RNAs (e.g. 5S rRNA, tRNA and miRNA) were separated from high molecular-weight (HMW) nucleic acids (e.g. 28S and 18S rRNA, mRNA and genomic DNA) by fractionation using isopropanol. HMW RNAs precipitated in 20% isopropanol, while most LMW RNAs remained in the supernatant. The purified LMW RNAs were used successfully for RNA gel blotting to detect miRNAs expressed in Arabidopsis and tomato (Lycopersicon esculentum) seeds. To increase the detection sensitivity of the microRNA probes, additional digoxigenin-labelled uridine triphosphates (UTPs) were incorporated into the miRNA probes by designing template oligo DNAs with three extra adenines (A) at each end of their sequence. These DNA oligomers were used to make double-stranded DNA templates for miRNA probe synthesis. This probe (termed AAAPLUS) exhibited stronger signals than normal probes. A technique was also developed to quickly screen expressed miRNAs in seeds using a miniblot system, which enabled simultaneous examination with multiple miRNA probes. This method provides a simple alternative to microRNA microarrays to identify the major miRNAs expressed in seeds.

Copyright

Corresponding author

*Correspondence: Fax: +1 541 737 3479hiro.nonogaki@oregonstate.edu
Present address: National Forage Seed Production Research Center, USDA-ARS, 3450 SW Campus Way, Corvallis, OR 97331-7102, USA.

References

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Achard, P., Herr, A., Baulcombe, D.C. and Harberd, N.P. (2004) Modulation of floral development by a gibberellin-regulated microRNA. Development 131, 33573365.
Babak, T., Zhang, W., Morris, Q., Blencowe, B.J. and Hughes, T.R. (2004) Probing microRNAs with microarrays: tissue specificity and functional inference. RNA 10, 18131819.
Barad, O., Meiri, E., Avniel, A., Aharonov, R., Barzilai, A., Bentwich, I., Einav, U., Gilad, S., Hurban, P., Karov, Y., Lobenhofer, E.K., Sharon, E., Shiboleth, Y.M., Shtutman, M., Bentwich, Z. and Einat, P. (2004) MicroRNA expression detected by oligonucleotide microarrays: System establishment and expression profiling in human tissues. Genome Research 14, 24862494.
Bartel, B. and Bartel, D.P. (2002) MicroRNAs: at the root of plant development?. Plant Physiology 132, 709717.
Calin, G.A., Liu, C.G., Sevignani, C., Ferracin, M., Felli, N., Dumitru, C.D., Shimizu, M., Cimmino, A., Zupo, S., Dono, M., Dell'Aquila, M.L., Alder, H., Rassenti, L., Kipps, T.J., Bullrich, F., Negrini, M. and Croce, C.M. (2004) MicroRNA profiling reveals distinct signatures in B cell chronic lymphocytic leukemias. Proceedings of the National Academy of Sciences, USA 101, 1175511760.
Carrington, J.C. and Ambros, V. (2003) Role of microRNAs in plant and animal development. Science 301, 336338.
Chen, F., Nonogaki, H. and Bradford, K.J. (2002) A gibberellin-regulated xyloglucan endotransglycosylase gene is expressed in the endosperm cap during tomato seed germination. Journal of Experimental Botany 53, 215223.
Chen, X.M. (2004) A microRNA as a translational repressor of APETALA2 in Arabidopsis flower development. Science 303, 20222025.
Engler-Blum, G., Meier, M., Frank, J. and Muller, G.A. (1993) Reduction of background problems in nonradioactive Northern and Southern blot analyses enables higher sensitivity than 32 P-based hybridizations. Analytical Biochemistry 210, 235244.
Floyd, S.K. and Bowman, J.L. (2004) Gene regulation: ancient microRNA target sequences in plants. Nature 428, 485486.
Juarez, M.T., Kui, J.S., Thomas, J., Heller, B.A., Timmermans, M.C.P. (2004) microRNA-mediated repression of rolled leaf1 specifies maize leaf polarity. Nature 428, 8488.
Krueger, S.K. and Williams, D.E. (1995) Quantitation of digoxigenin-labeled DNA hybridized to DNA and RNA slot blots. Analytical Biochemistry 229, 162169.
Laufs, P., Peaucelle, A., Morin, H. and Traas, J. (2004) MicroRNA regulation of the CUC genes is required for boundary size control in Arabidopsis meristems. Development 131, 43114322.
Llave, C., Kasschau, K.D., Rector, M.A. and Carrington, J.C. (2002a) Endogenous and silencing-associated small RNAs in plants. Plant Cell 14, 16051619.
Llave, C., Xie, Z., Kasschau, K.D. and Carrington, J.C. (2002b) Cleavage of Scarecrow-like mRNA targets directed by a class of Arabidopsis miRNA. Science 297, 20532056.
McHale, N.A. and Koning, R.E. (2004b) MicroRNA-directed cleavage of Nicotiana sylvestris PHAVOLUTA mRNA regulates the vascular cambium and structure of apical meristems. Plant Cell 16, 17301740.
Miska, E.A., Alvarez-Saavedra, E., Townsend, M., Yoshii, A., Sestan, N., Rakic, P., Constantine-Paton, M. and Horvitz, H.R. (2004) Microarray analysis of microRNA expression in the developing mammalian brain. Genome Biology 5 R68
Nath, U., Crawford, B.C.W., Carpenter, R. and Coen, E. (2003) Genetic control of surface curvature. Science 299, 14041407.
Nonogaki, H. and Bradford, K.J. (2003) Tissue printing for localization of mRNA expression in seeds. pp. in 171179. Nicolás, G.;, Bradford, K.J.;, Côme, D.;, Pritchard, H.W. (Eds) The biology of seeds: Recent research advances. Wallingford, CAB International.
Nonogaki, H., Gee, O.H. and Bradford, K.J. (2000) A germination-specific endo-β-mannanase gene is expected in the micropylar endosperm cap of tomato seeds. Plant Physiology 123, 12351245.
Palatnik, J.F., Allen, E., Wu, X., Schommer, C., Schwab, R., Carrington, J.C. and Weigel, D. (2003) Control of leaf morphogenesis by microRNAs. Nature 425, 257263.
Penfield, S., Meissner, R.C., Shoue, D.A., Carpita, N.C. and Bevan, M.W. (2001) MYB61 is required for mucilage deposition and extrusion in the Arabidopsis seed coat. Plant Cell 13, 27772791.
Poy, M.N., Eliasson, L., Krutzfeldt, J., Kuwajima, S., Ma, X., MacDonald, P.E., Pfeffer, B., Tuschl, T., Rajewsky, N., Rorsman, P. and Stoffel, M. (2004) A pancreatic islet-specific microRNA regulates insulin secretion. Nature 432, 226230.
Reinhart, B.J., Weinstein, E.G., Rhoades, M.W., Bartel, B. and Bartel, D.P. (2002) MicroRNAs in plants. Genes and Development 16, 16161626.
Rhoades, M.W., Reinhart, B.J., Lim, L.P., Burge, C.B., Bartel, B. and Bartel, D.P. (2002) Prediction of plant microRNA targets. Cell 110, 513520.
Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular cloning: A laboratory manual. 2nd editionNew York, Cold Spring Harbor Laboratory Press.
Sunkar, R. and Zhu, J.K. (2004) Novel and stress-regulated microRNAs and other small RNAs from Arabidopsis. Plant Cell 16, 20012019.
Wu, C.-T., Leubner-Metzger, G., Meins, F. and Bradford, K.J. (2001) Class I β-1,3-glucanase and chitinase are expressed in the micropylar endosperm of tomato seeds prior to radicle emergence. Plant Physiology 126, 12991313.

Keywords

Simple purification of small RNAs from seeds and efficient detection of multiple microRNAs expressed in Arabidopsis thaliana and tomato (Lycopersicon esculentum) seeds

  • Ruth C. Martin (a1), Po-Pu Liu (a1) and Hiroyuki Nonogaki (a1)

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