Optimization of RNA Extraction Method of Banana Leaf Using Li Buffer and GENEzol Reagent

Ida Ayu Mas Amelia Kusumaningtyas(1*), Arief Pambudi(2), Yunus Effendi(3),


(1) Universitas Al-Azhar Indonesia
(2) Universitas Al-Azhar Indonesia
(3) Universitas Al-Azhar Indonesia
(*) Corresponding Author

Abstract


High quality extracted RNA from plant tissues is used in downstream transcriptomic application that needs high quantity for multiple analysis. Attaining high quality and high yield for banana plant tissue is a challenge with its high number of secondary metabolites becoming contaminants, effecting the purity of RNA extracted. A procedure to extract banana plant RNA with high quality and quantity in this study is developed to be simple, robust, affordable and accessible using basic lab equipment and materials. This study optimizes RNA extraction from the leaf of juvenile Pisang Susu (Musa spp.) using a combination of Li buffer (modified CTAB) and GENEzol reagent. Treatment differs in the use of β-mercaptoethanol and the duration of sample precipitation with isopropanol. NucleoSpin RNA Plant extraction kit was made as comparison. Measurement of RNA quantity used quantus fluorometer, and quality measured by spectrophotometer. Results showed that the addition of β-mercaptoethanol in Li buffer is vital with samples left to precipitate overnight providing best results. The total RNA obtained had a higher yield compared to the commercial kit with 108 – 211 ng/µl and 0.35 – 0.37 ng/µl respectively. Light absorbance A260/280 indicating sample purity between method has similar RNA quality of 0.917 – 1.084 and 0.843 – 1.026. This study proved that modification of CTAB buffer using Li buffer for RNA extraction resulted in better quality and quantity of RNA compared to the commercial RNA extraction kit.

Keywords – Banana, Optimization, RNA Extraction.


Full Text:

PDF

References


Z. Feng, Y. Mao, N. Xu, B. Zhang, P. Wei, D.-L. Yang, Z. Wang, Z. Zhang, R. Zheng, L. Yang, L. Zeng, X. Liu and J.-K. Zhu, "Multigeneration Analysis Reveals the Inheritance, Specificity, and Patterns of CRISPR/Cas-Induced Gene Modifications in Arabidopsis," The Proceedings of the National Academy of Sciences, vol. 111, no. 12, pp. 4632-4637, 2014.

S. Fleige and M. W. Pfaffl, "RNA Integrity and The Effect on The Real-Time qRT-PCR Performance," Molecular Aspects of Medicine, vol. 27, pp. 126-139, 2006.

C. Becker, A. Hammerle-Fickinger, I. Riedmaier and M. W. Pfaffl, "mRNA and microRNA Quality Control for RT-qPCR Analysis," Methods, vol. 50, no. 4, pp. 237-243, 2010.

S. Taylor, M. Wakem, G. Dijkman, M. Alsarraj and M. Nguyen, "A Practical Approach to RT-qPCR-Publishing Data that Conform to the MIQE Quidelines," Methods, vol. 50, no. 4, pp. S1-5, 2010.

D. Alpern, V. Gardeux, J. Russeil, B. Mangeat, A. C. A. Meireles-Filho, R. Breysse, D. Hacker and B. Deplancke, "BRB-seq: Ultra-Affordable High-throughput Transcriptomics Enabled by Bulk RNA Barcoding and Sequencing," Genome Biology, vol. 20, no. 71, 2019.

M. T. Hidayat and E. N. Prasetyo, "Modified Spin Column-Based RNA Extraction Methods of Staphylococcus aureususing PureLink®RNA Mini Kit and Basic Laboratory Instrument," Indonesian Journal of Medical Laboratory Science and Technology, vol. 3, no. 2, p. 73–80, 2021.

R. Yockteng, A. M. R. Almeida, S. Yee, T. Andre, C. Hill and C. D. Specht, "A Method for Extracting High-Quality RNA from Diverse Plants for Next-Generation Sequencing and Gene Expression Analyses," Applications in Plant Sciences, vol. 1, no. 2, 2013.

M. Kaushal, G. Mahuku and R. Swennen, "Comparative Transcriptome and Expression Profiling of Resistant and Susceptible Banana Cultivars during Infection by Fusarium oxysporum," International Journal of Molecular Sciences, vol. 22, no. 6, 2021.

L. Zhang, A. Cenci, M. Rouard, D. Zhang, Y. Wang, W. Tang and Si-JunZheng, "Transcriptomic Analysis of Resistant and Susceptible Banana Corms in Response to Infection by Fusarium oxysporum f. sp. cubense Tropical Race 4," Scientific Reports, vol. 9, no. 8199, 2019.

M. Drapal, E. B. d. Carvalho, M. Rouard, D. Amah, J. Sardos, I. V. d. Houwe, A. Brown, N. Roux, R. Swennen and P. D. Fraser, "Metabolite Profiling Characterises Chemotypes of Musa Diploids and Triploids at Juvenile and Pre-Flowering Growth Stages," Scientific Reports, vol. 9, no. 4657, 2019.

M. H. Asif, P. Dhawan and P. Nath, "A Simple Procedure for the Isolation of High Quality RNA from Ripening Banana Fruit," Plant Molecular Biology Reporter, vol. 18, pp. 109-115, 2000.

S. Zulaeha, D. Purwoko, I. Cartealy, T. Tajuddin, Karyanti and H. Khairiyah, "Comparison of Three RNA Extraction Kits for Transcriptome Analysis of Oil Palm (Elaeis guineensis Jacq.)," Jurnal Bioteknologi dan Biosains Indonesia, vol. 6, no. 1, pp. 118-129, 2019.

A. R. Vennapusa, I. M. Somayanda, C. J. Doherty and S. V. K. Jagadish, "A Universal Method for High-Quality RNA Extraction from Plant Tissues Rich in Starch, Proteins and Fiber," Scientific Reports, vol. 10, no. 16887, 2020.

A. C. Da Luz, I. R. Pretti and M. D. C. P. Batitucci, "Comparison of RNA Extraction Methods for Passiflora edulis Sims Leaves," Revista Brasileira de Fruticultura, vol. 38, no. 1, 2016.

K. Nugroho, D. Satyawan, I. M. Tasma and P. Lestari, "Genomic DNA Extraction: The Critical Stage in Plant Molecular Analysis Activities," AgroBiogen, vol. 18, no. 1, pp. 33-44, 2022.

M. E. Haque, L. Marriott, N. Naeem, T. Henry, A. J. Conde and M. Kersaudy-Kerhoas, "A Low-Cost, Open-Source Centrifuge Adaptor for Separating Large Volume Clinical Blood Samples," Plos One, vol. 17, no. 7, 2022.

D. Mbéguié-A-Mbéguié, B. Fils-Lycaon, M. Chillet, O. Hubert, C. Galas and R.-M. Gomez, "Extraction and Purification of Total RNA from Banana Tissues (Small Scale)," Fruits, vol. 63, no. 4, pp. 255-261, 2008.

C. Li, J. Shao, Y. Wang, W. Li, D. Guo, B. Yan, Y. Xia and M. Peng, "Analysis of Banana Transcriptome and Global Gene Expression Profiles in Banana Roots in Response to Infection by Race 1 and Tropical Race 4 of Fusarium oxysporum f. sp. cubense," BMC Genomics, vol. 14, no. 851, 2013.

Y. Effendi, Miftahudin and A. Pambudi, "Respons Molekuler Sistem Pertahanan Tanaman Pisang (Musa spp.) terhadap Serangan Bakteri Patogen Ralstonia solanacearum Penyebab Penyakit Layu Bakteri," Universitas Al Azhar Press, Jakarta, 2018.




DOI: http://dx.doi.org/10.36722/sst.v10i2.2092

Refbacks

  • There are currently no refbacks.


LP2M (Lembaga Penelitian dan Pengembangan Masyarakat)

Universitas AL-AZHAR INDONESIA, Lt.2 Ruang 207

Kompleks Masjid Agung Al Azhar

Jl. Sisingamangaraja, Kebayoran Baru

Jakarta Selatan 12110

Visitor

 This work is licensed under CC BY 4.0