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Rapid and Reliable Method of High-Quality RNA Extraction from Diverse Plants

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http://www.scirp.org/journal/PaperInformation.aspx?PaperID=50398#.VDzCwVfHRK0

The isolation of high quality RNA is a crucial technique in plant molecular biology. The quality of RNA determines the reliability of downstream process like real time PCR. In this paper, we reported a high quality RNA extraction protocol for a variety of plant species. Our protocol is time effective than traditional RNA extraction methods. The method takes only an hour to complete the procedure. Spectral measurement and electrophoresis were used to demonstrate RNA quality and quantity. The extracted RNA was further used for cDNA synthesis, expression analysis and copy number determination through Real Time PCR. The results indicate that RNA was of good quality and fit for real time PCR. This high throughput plant RNA extraction protocol can be used to isolate high quality RNA from diverse plants for real time PCR and other downstream applications.
Cite this paper
Sah, S. , Kaur, G. and Kaur, A. (2014) Rapid and Reliable Method of High-Quality RNA Extraction from Diverse Plants. American Journal of Plant Sciences, 5, 3129-3139. doi: 10.4236/ajps.2014.521329. 
 

[1] Yockteng, R., Almeida, A.M.R., Yee, S., Andre, T., Hill, C. and Specht, C.D. (2013) A Method for Extracting High-Quality RNA from Diverse Plants for Next Generation Sequencing and Gene Expression Analysis. Application in Plant Science, 1, Article ID: 13000700. http://dx.doi.org/10.3732/apps.1300070
[2] MacRae, E. (2007) Extraction of Plant RNA. In: Hilario, E. and Mackay, J., Eds., Methods in Molecular Biology, 353, 15-24. Protocols for Nucleic Acid Analysis by Nonradioactive Probes, 2nd Edition, Humana Press, Totowa.
[3] Loomis, W.D. (1974) Overcoming Problems of Phenolics and Quinines in the Isolation of Plant Enzymes and Organelles. Methods in Enzymology, 131, 528-544. http://dx.doi.org/10.1016/0076-6879(74)31057-9
[4] Logemann, J., Schell, J. and Wilmitzer, L. (1987) Improvement Method for the Isolation of RNA from Plant Tissues. Analytical Biochemistry, 163, 45-50. http://dx.doi.org/10.1016/0003-2697(87)90086-8
[5] Dabo, S.M., Michell Jr., E.D. and Melcher, U.A. (1993) A Method for the Isolation of Nuclear DNA from Cotton (Gossypium) Leaves. Analytical Biochemistry, 210, 34-38.
http://dx.doi.org/10.1006/abio.1993.1146
[6] Asif, M.H., Dhawan, P. and Nath, P. (2000) A Simple Procedure for Isolation of High Quality RNA from Ripening Banana Fruit. Plant Molecular Biology Reporter, 18, 109-115.
http://dx.doi.org/10.1007/BF02824018
[7] Ky, H., Yeap, S.K. and Napis, S.B. (2012) The Best Method for Isolated Total RNA from Durian Tissues. International Food Research Journal, 19, 1181-1183.
[8] Ghawana, S., Paul, A., Kumar, H., Kumar, A., Singh, H., Bhardwaj, P.K. and Rani, A., et al. (2011) An RNA Isolation System for Plant Tissues Rich in Secondary Metabolites. BMC Research Notes, 4, 85.
http://dx.doi.org/10.1186/1756-0500-4-85
[9] Vasanthaiah, H.K.N., Ketam, R. and Sheikh, M.B. (2008) Efficient Protocol for Isolation of Functional RNA from Different Grape Tissue Rich in Polyphenols and Polysaccharides for Gene Expression Studies. Electronic Journal of Biotechnology, 11. http://dx.doi.org/10.2225/vol11-issue3-fulltext-5
http://www.ejbiotechnology.cl/content/vol11/issue3/full/5/index.html. ISSN 0717-3458
[10] Gehrig, H.H., Winter, K., Cushman, J., Borland, A. and Taybi, T. (2000) An Improved RNA Isolation Method for Succulent Plant Species Rich in Polyphenols and Polysaccharides. Plant Molecular Biology Reporter, 18, 369-376.
http://dx.doi.org/10.1007/BF02825065
[11] Meng, L. and Feldman, L. (2010) A Rapid TRIzol-Based Two-Step Method for DNA-Free RNA Extraction from Arabidopsis Siliques and Dry Seeds. Biotechnology Journal, 5, 183-186. http://dx.doi.org/10.1002/biot.200900211
[12] Qi, G., Li, J.T., Ruan, Q.P., Yang, J. and Su, Z.X. (2009) An Optimized, Small-Scale Preparation of High-Quality RNA from Dry Seeds of Davidia involucrata. Phytochemical Analysis, 20, 139-142.
http://dx.doi.org/10.1002/pca.1108
[13] Berendzen, K., Searle, I., Ravenscroft, D., Koncz, C., Batschauer, A., Coupland, G., Somssich, I.E. and Ulker, B. (2005) A Rapid and Versatile Combined DNA/RNA Extraction Protocol and Its Application to the Analysis of a Novel DNA Marker Set Polymorphic between Arabidopsis thaliana Ecotypes Col-0 and Landsberg erecta. Plant Methods, 1, 4.
http://dx.doi.org/10.1186/1746-4811-1-4
[14] Kumar, A. and Singh, K. (2012) Isolation of High Quality RNA from Phyllanthus emblica and Its Evaluation by Downstream Applications. Molecular Biotechnology, 52, 269-275. http://dx.doi.org/10.1007/s12033-011-9492-5
[15] Gudenschwager, O., González-Agüero, M. and Defilippi, B.G. (2012) A General Method for High-Quality RNA Isolation from Metabolite-Rich Fruits. South African Journal of Botany, 83, 186-192.
http://dx.doi.org/10.1016/j.sajb.2012.08.004
[16] Japelaghi, R.H., Haddad, R. and Garoosi, G.A. (2011) Rapid and Efficient Isolation of High Quality Nucleic Acids from Plant Tissues Rich in Polyphenols and Polysaccharides. Molecular Biotechnology, 49, 129-137.
http://dx.doi.org/10.1007/s12033-011-9384-8
[17] Yin, D., Liu, H., Zhang, X. and Cui, D. (2011) A Protocol for Extraction of High-Quality RNA and DNA from Peanut Plant Tissues. Molecular Biotechnology, 49, 187-191. http://dx.doi.org/10.1007/s12033-011-9391-9
[18] Box, M.S., Coustham, V., Dean, C. and Mylne, J.S. (2011) Protocol: A Simple Phenol-Based Method for 96 Well Extraction of High Quality RNA from Arabidopsis. Plant Methods, 7, 7. http://dx.doi.org/10.1186/1746-4811-7-7
[19] Maciel, B.M., Dias, J.C., Romano, C.C., Sriranganathan, N., Brendel, M. and Rezende, R.P. (2011) Detection of Salmonella enteritidis Is Asymptomatic Carrier Animals: Comparison of Quantitative Real Time PCR and Bacteriological Culture Methods. Genetics and Molecular Research, 10, 2578-2588.
[20] Chomczynski, P. and Sacchi, N. (1987) Single-Step Method of RNA Isolation by Acid Guanidinium Thiocyanate-Phenol-Chloroform Extraction. Analytical Biochemistry, 162, 156-159.
http://dx.doi.org/10.1016/0003-2697(87)90021-2
[21] Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. 2nd Edition, Cold Spring Harbor Lab., Cold Spring Harbor, New York.
[22] Rohland, N. and Reich, D. (2012) Cost-Effective, High-Throughput DNA Sequencing Libraries for Multiplexed Target Capture. Genome Research, 22, 939-946.
http://dx.doi.org/10.1101/gr.128124.111
[23] Singh, K., Raizada, J., Bhardwaj, P., Ghawana, S., Rani, A., Singh, H., Kaul, K. and Kumar, S. (2004) 26S rRNA-Based Internal Control Gene Primer Pair for Reverse Transcription-Polymerase Chain Reaction-Based Quantitative Expression Studies in Diverse Plant Species. Analytical Biochemistry, 335, 330-333.
http://dx.doi.org/10.1016/j.ab.2004.08.030
[24] Jain, M., Tyagi, A.K. and Khurana, J.P. (2006) Molecular Characterization and Differential Expression of Cytokinin-Responsive Type—A Response Regulators in Rice (Oryza sativa). BMC Plant Biology, 6, 1.
http://dx.doi.org/10.1186/1471-2229-6-1
[25] Iskandar, H.M., Simpson, R.S., Casu, R.E., Bonnett, G.D., Maclean, D.J. and Manners, J.M. (2004) Comparison of Reference Genes for Quantitative Real-Time Polymerase Chain Reaction Analysis of Gene Expression in Sugarcane. Plant Molecular Biology Reporter, 22, 325-337. http://dx.doi.org/10.1007/BF02772676
[26] Goidin, D., Mamessier, A., Staquet, M.J., Schmitt, D. and Berthier-Vergnes, O. (2001) Ribosomal 18S RNA Prevails over Glyceraldehydes-3-Phosphate Dehydrogenase and Beta-Actin Genes as Internal Standard for Quantitative Comparison of mRNA Levels in Invasive and Noninvasive Human Melanoma Cell Subpopulations. Analytical Biochemistry, 295, 17-21. http://dx.doi.org/10.1006/abio.2001.5171
[27] Ginzinger, D.G. (2002) Gene Quantification Using Real-Time Quantitative PCR: An Emerging Technology Hits the Mainstream. Experimental Hematology, 30, 503-512. http://dx.doi.org/10.1016/S0301-472X(02)00806-8
[28] Hindson, B.J., Ness, K.D., Masquelier, D.A., Belgrader, P., Heredia, N.J., Colston, B.W., et al. (2011) High-Through-put Droplet Digital PCR System for Absolute Quantitation of DNA Copy Number. Analytical Chemistry, 83, 8604-8610.
http://dx.doi.org/10.1021/ac202028g
[29] Cankar, K., Stebih, D., Dreo, T., Zel, J. and Gruden, K. (2006) Critical Points of DNA Quantification by Real-Time PCR-Effects of DNA Extraction Method and Sample Matrix on Quantification of Genetically Modified Organisms. BMC Biotechnology, 6, 37. http://dx.doi.org/10.1186/1472-6750-6-37                                           eww141014lx
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