An Efficient Protocol for Total RNA Isolation from Healthy and Stressed Tissues of Mulberry (Morus sp.) and Other Species

Radha Sivarajan Sajeevan, Manchanahally Byrappa Shivanna, Karaba N. Nataraja

Department of Crop Physiology, University of Agricultural Sciences, GKVK, Bangalore, India.
Department of Crop Physiology, University of Agricultural Sciences, GKVK, Bangalore, India Department of Studies in Applied Botany, Kuvempu University, Shankaraghatta, Shimoga District, India.
Department of Studies in Applied Botany, Kuvempu University, Shankaraghatta, Shimoga District, India.

DOI: 10.4236/ajps.2014.513221                

Abstract

Extraction of quality RNA for molecular biology applications from perennial woody plants like mulberry is complicated due to the presence of high polysaccharides, polyphenols and other secondary metabolites. Since the existing methods failed to yield quality RNA in sufficient quantity from leaf and root tissues of mulberry, in this study, we modified the CTAB-based protocol. The standardised protocol yielded high quantity (520.00 μg/g fresh weight of leaf tissue) of quality RNA and the RNA extracted was suitable for all downstream applications such as cDNA synthesis, PCR and whole transcriptome analysis. The method developed was also found to be useful for isolating good quality and quantity total RNA from desiccated and salinity stressed leaf tissues of mulberry. The protocol was also applied successfully to isolate total RNA from leaf tissues of other species such as cardamom, papaya and rice.

Keywords

Mulberry, Cardamom, Papaya, Rice, Dehydration, Salinity, RNA Isolation

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	Gu, X.D., Sun, M.Y., Zhang, L., Fu, H.W., Cui, L., Chen, R.Z., Zhang, D.W. and Tian, J.K. (2010) UV-B Induced Changes in the Secondary Metabolites of Morus alba L. Leaves. Molecules, 15, 2980-2993. http://dx.doi.org/10.3390/molecules15052980
[2]	Chan, K.C., Ho, H.H., Huang, C.N., Lin, M.C., Chen, H.M. and Wang, C.J. (2009) Mulberry Leaf Extract Inhibits Vascular Smooth Muscle Cell Migration Involving a Block of Small GTPase and Akt/NF-kB Signals. Journal of Agricultural and Food Chemistry, 57, 9147-9153. http://dx.doi.org/10.1021/jf902507k
[3]	Memon, A.A., Memon, N., Luthria, L.D.L., Bhanger, M L. and Pitafi, A.A. (2010) Phenolic Acids Profiling and Antioxidant Potential Of Mulberry (Morus laeviagata, Morus nigra L., Morus alba L.) Leaves and Fruits Grown in Pakistan. Polish Journal of Food and Nutrition Sciences, 60, 25-32.
[4]	McMurry, J. (1992) Organic Chemistry. Brooks/Cole, California, 1021-1023.
[5]	Salzman, R.A., Fujita, T., Zhu-Salzman, K., Hasegawa, P.M. and Bressan, R.A. (1999) An Improved RNA Isolation Method for Plant Tissues Containing High Levels of Phenolic Compounds or Carbohydrates. Plant Molecular Biology Reporter, 17, 11-17. http://dx.doi.org/10.1023/A:1007520314478
[6]	Sharma, P., Jha, A.B., Dubey, R.S. and Pessarakli, M. (2012) Reactive Oxygen Species, Oxidative Damage, and Antioxidative Defense Mechanism in Plants under Stressful Conditions. Journal of Botany, 2012, Article ID: 217037, 26 p. http://dx.doi.org/10.1155/2012/217037
[7]	Logemann, J., Schell, J. and Willmitzer, L. (1987) Improved Method for the Isolation of RNA from Plant Tissues. Analytical Biochemistry, 163, 16-20. http://dx.doi.org/10.1016/0003-2697(87)90086-8
[8]	Gasic, K., Hernandez, A. and Korban, S.S. (2004) RNA Extraction from Different Apple Tissues Rich in Polyphenols and Polysaccharides for cDNA Library Construction. Plant Molecular Biology Reporter, 22, 437-437. http://dx.doi.org/10.1007/BF02772687
[9]	Karaba, A., Dixit, S., Greco, R., Aharoni, A., Trijatmiko, K.R., Marsch-Martinez, N., Krishnan, A., Nataraja, K.N., Udayakumar, M. and Pereira, A. (2007) Improvement of Water Use Efficiency in Rice by Expression of HARDY, an Arabidopsis Drought and Salt Tolerance Gene. Proceedings of the National Academy of Sciences of United States of American, 104, 15270-15275. http://dx.doi.org/10.1073/pnas.0707294104
[10]	Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. 3rd Edition, Cold Spring Harbor Laboratory Press, New York.
[11]	Lambert, J.D., Chan, X.Y., Spiecker, B. and Sweet, H.C. (2010) Characterizing the Embryonic Transcriptome of the Snail Ilyanassa. Integrative and Comparative Biology, 50, 768-777. http://dx.doi.org/10.1093/icb/icq121
[12]	Hughes, D.W. and Galau, G. (1988) Preparation of RNA from Cotton Leaves and Pollen. Plant Molecular Biology Reporter, 6, 253-257. http://dx.doi.org/10.1007/BF02670385
[13]	Sharma, A.D., Gill, P.K. and Singh, P. (2003) RNA Isolation from Plant Tissues Rich in Polysaccharides. Analytical Biochemistry, 314, 319-321. http://dx.doi.org/10.1016/S0003-2697(02)00689-9
[14]	Fu, X., Shulin, D., Guohua, S., Qinglu, Z. and Suhua, S. (2004) Isolating High-Quality RNA from Mangroves without Liquid Nitrogen. Plant Molecular Biology Reporter, 22, 197a-197e. http://dx.doi.org/10.1007/BF02772728
[15]	Zeng, H.C., Deng, L.H. and Zhang, C.F. (2006) Cloning of Salt Tolerance-Related cDNAs from the Mangrove Plant Sesuvium portulacastrum L. Journal of Integrative Plant Biology, 48, 952-957. http://dx.doi.org/10.1111/j.1744-7909.2006.00287.x
[16]	Yang, G., Zhou, R., Tang, T. and Shi, S. (2008) Simple and Efficient Isolation of High-Quality Total RNA from Hibiscus tiliaceus, a Mangrove Associate and Its Relatives. Preparative Biochemistry and Biotechnology, 38, 257-264. http://dx.doi.org/10.1080/10826060802164991
[17]	Vasanthaiah, H.K.N., Katam, 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, 5. http://dx.doi.org/10.2225/vol11-issue3-fulltext-5
[18]	Rubio-Piña, J.A. and Vázquez-Flota, F.A. (2008) Isolation of Functional Total RNA from Argemone mexicana Tissues. Electronic Journal of Biotechnology, 11, 13. http://dx.doi.org/10.2225/vol11-issue4-fulltext-13
[19]	Wallace, D.M. (1987) Large and Small-Scale Phenol Extractions. Methods Enzymology, 152, 33-41. http://dx.doi.org/10.1016/0076-6879(87)52007-9
[20]	Srivastava, N., Chaudhary, S., Kumar, V., Katudia, K., Vaidya, K., Vyas, M.K. and Chikara, S.K. (2012) Evaluation of the Yield, Quality and Integrity of Total RNA Extracted by Four Different Extraction Methods in Rice (Oryza sativa). Journal of Crop Science and Technology, 1, 1-9.
[21]	Morante-Carriel, J., Sellés-Marchart, S., Martínez-Márquez, A., Martínez-Esteso, M.J., Luque, I. and Bru-Martínez, R. (2014) RNA Isolation from Loquat and Other Recalcitrant Woody Plants with High Quality and Yield. Analytical Biochemistry, 452, 46-53. http://dx.doi.org/10.1016/j.ab.2014.02.010