Innovative and Frugal Protocols for RNA Related Experiments

Author(s)

Halagappa Eshwarappa Shashidhar , Jameel Ahmad Khan , Baba Fakrudin , Edries Yousaf Hajam ,

Download Full PDF Pages: 123-131 | Views: 327 | Downloads: 124 | DOI: 10.5281/zenodo.3529430

Volume 3 - October 2019 (10)

Abstract

Rice is a staple crop and is being cultivated far and wide around the globe. The waterloggedgged habit of cultivation has made rice extremely sensitive to drought. Understanding expression profiles will open new avenues of research for drought tolerance in rice. The presentsent study aims at utilizing the most innovative and frugal ways for highly sensitive molecular biology protocols. These protocols will help researchers to undertake RNA work in any lab with basic amenities. A details methodologies with critical tips are compiled and presented here for RNA extraction, RNA detection and finally expression profiles of two genotypes. This proof of concept for novel methods is a complete guide for beginner in RNA work with minute detailing of each step

Keywords

Bleach protocol, Drought, Primerdimer, Rice, RNA, RNase, qPCR.

References

         i.        Babu, R. C., Zhang, J., Blum, A., Ho, T. H. D., Wu, R. and Nguyen, H. T. 2016. HVA1 , a LEA Gene from Barley Confers Dehydration Tolerance in Transgenic Rice (Oryza Sativa L .) via Cell Membrane Protection . Plant Sci. 166(4):855-62.

ii.      Ball, T. B., Plummer, F. A. and HayGlass, K. T. 2003. Improved MRNA Quantitation in LightCycler RT-PCR. International Archives of Allergy and Immunology 3: 82–86.

iii.    Chomczynski, P, and N Sacchi. 1987. Single Step Method of RNA Isolation by Acid Guanidinium Thiocyanate-Phenol Cloroform Extraction.Anal Biochem. 162: 156–59.

iv.     Dalaly, B. K., Eitenmiller, R. R., Friend, B. A. and Shahani, K. M. 1980. Human Milk Ribonuclease.Biochimica et Biophysica Acta (BBA)-Enzymology. 5(5858): 381–91.

v.       Gachon, C., Mingam, A. and Charrier, B. 2004. Real-Time PCR: What Relevance to Plant Studies? ´. Journal of Experimental Botany. 55(402): 1445–54.

vi.     Harder, J. and Schröder, J. M. 2002. RNase 7 , a Novel Innate Immune Defense Antimicrobial Protein of Healthy Human Skin. Ournal of Biological Chemistry. 277(48): 46779–84.

vii.   Kalnitsky, G. and Resnick, H. 1959. The Effect of an Altered Secondary Structure on Ribonuclease Activity. Ournal of Biological Chemistry. 234 (7): 1714-1717.

viii. Khan, J. A. and Shashishar H. E. 2018. A Novel High Throughput Root Phenotyping Protocol and Structure Using Lemna Tec 3D. Mysore J. Agric. Sci. 52 (2): 356–68.

ix.     Khan, J. A., Krupa, K. N.,Holla, S. K. M., Sowjanya, M. S., Dar, Z. A., and Shashidhar, H. E., 2018. Micronutrient Productivity: A Comprehensive Parameter for Biofortification in Rice ( Oryza Sativa L.) Grain. Journal of the Science of Food and Agriculture. 99(3): 1311-132.

x.       Aranda, P. S., LaJoie, D. M., & Jorcyk, C. L. 2012. Bleach gel: a simple agarose gel for analyzing RNA quality. Electrophoresis, 33(2): 366-369.

xi.     Livak, K. J., and Schmittgen, T. D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method. methods, 25(4): 402-408.

xii.   Shashidhar, H. E., Kanbar, A., Toorchi, M., Raveendra, G. M., Kundur, P., Vimarsha, H. S., Soman, R., Kumar, N. G., Bekele, B. D. and Bhavani, P., 2013. Breeding for drought resistance using whole plant architecture—conventional and molecular approach. Plant breeding from laboratories to Fields. Vienna, Austria.

xiii. Steele, K. A., Price, A. H., Shashidhar, H. E., and Witcombe, J. R. 2006. Marker-assisted selection to introgress rice QTLs controlling root traits into an Indian upland rice variety. Theoretical and Applied Genetics, 112(2): 208-221.

xiv. VVarma, C. M. K., Gouda, P. K., Saikumar, S., Shenoy, V., Shashidhar, H. E., and Neelamraju, S. 2012. Transgressive segregation for yield traits in Oryza sativa IR58025B X Oryza meridionalis Ng. Bc 2 F 3 population under irrigated and aerobic conditions. Journal of Crop Science and Biotechnology, 15(3): 231-238..

xv.   Wang, S., Li, S., Liu, Q., Wu, K., Zhang, J., Wang, S., Wang, Y., Chen, X., Zhang, Y., Gao, C. and Wang, F., 2015. The OsSPL16-GW7 regulatory module determines grain shape and simultaneously improves rice yield and grain quality. Nature genetics, 47(8): 949..

          xvi. Yin, C. C., Ma, B., Wang, W., Xiong, Q., Zhao, H., Chen, S. Y. and Zhang, J. S. 2016. “RNA Extraction and Preparation in Rice ( Oryza Sativa ). Current protocols in plant biology. 1: 411–418.

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