Abstract
For the experimental analysis of miRNAs and other small RNAs in the 20–25 nucleotide (nt) size range, the first and most important step is the isolation of high-quality total RNA. Because RNA degradation products can mask or dilute the presence of true miRNAs, it is important when choosing a method that it efficiently extracts RNA from tissues in a manner that prevents degradation of RNA of both high and low molecular weight. In addition, the presence of polyphenols, polysaccharides, and secondary metabolites may render nucleic acids insoluble, and hinder the recovery of the miRNAs. Finally, and most importantly, the method chosen must be capable of retaining the small RNA component. In this chapter, we will present a set of total RNA isolation methods that can be used to maximize the recovery of high-quality RNA to be used in miRNA analysis for a large number of plant species and tissue types.
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References
Geuna F, Hartings H, Scienza A (1998) A new method for rapid extraction of high quality RNA from recalcitrant tissues in grapevine. Plant Mol Biol Rep 16:61–67
Salzman RA, Fugita T, Zhu-Salzman K, Haswgawa PM, Bressman RA (1999) An improved RNA isolation method for plant tissues containing high levels of phenolic compound or carbohydrates. Plant Mol Biol Rep 17:11–17
Tai HH, Pelletier C, Beardmore T (2004) Total RNA isolation from Pica mariana dry seed. Plant Mol Biol Rep 22:93a–93e
Yeh K, Juang R, Su J (1991) A rapid and efficient method for RNA isolation from plant with high carbohydrate content. Focus 13:102–103
Wallace DM (1987) Precipitation of Nucleic Acids. Meth Enzymol 152:41–48
Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159
Chomczynski P, Sacchi N (2006) The single-step method of RNA isolation by acid guanidinium sothiocyanate-phenol-chloroformextraction: wenty-something years on. Nat Protoc 1:581–585
Chomczynski P (1993) A reagent for the single-step simultaneous isolation of RNA, DNA, and proteins from cell and tissue samples. BioTechniques 15:532–537
Grahm GC (1993) A method of extracting total RNA from Pinus radiata and other conifers. Plant Mol Biol Rep 11:32–37
Siju S, Madhubala R, Bhat AI (2006) Sodium sulphite enhances RNA isolation and sensitivity of Cucumber mosaic virus detection by RT-PCR in black pepper. J Virol Methods 141:107–110
Bugos RC, Chiang VL, Zhang XH, Campbell ER, Podila GK, Campbell WH (1995) RNA isolation from plant tissues recalcitrant to extraction in guanidine. BioTechniques 19:734–737
Loomis WD (1974) Overcoming problems of phenolics and quinones in the isolation of plant enzymes and organelles. Meth Enzymol 31:528–544
Logemann J, Schell J, Willmitzer L (1987) Improved method for the isolation of RNA from plant tissues. Anal Biochem 163:16–20
Manning K (1990) Isolation of nucleic acids from plants by differential solvent precipitation. Anal Biochem 195:45–50
Acknowledgments
The methods described in this chapter were developed with support from NSF, USDA and DOE.
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Accerbi, M., Schmidt, S.A., De Paoli, E., Park, S., Jeong, DH., Green, P.J. (2010). Methods for Isolation of Total RNA to Recover miRNAs and Other Small RNAs from Diverse Species. In: Meyers, B., Green, P. (eds) Plant MicroRNAs. Methods in Molecular Biology, vol 592. Humana Press. https://doi.org/10.1007/978-1-60327-005-2_3
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DOI: https://doi.org/10.1007/978-1-60327-005-2_3
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