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An Optimised TRIzol-based Protocol for the Improvement of RNA Extraction Yield of Tomato Stem

Anis Afifah, Prachumporn Nounurai, Rejeki Siti Ferniah, Hermin Pancasakti Kusumaningrum, Dyah Wulandari and Anto Budiharjo

Pertanika Journal of Tropical Agricultural Science, Volume 44, Issue 3, August 2021


Keywords: RNA extraction, RT-PCR, Solanum lycopersicum, TRIzol

Published on: 30 August 2021

One of the most common methods for purifying RNA is using TRIzol reagent because of its simplicity and economic feasibility. However, the drawback of this method is frequently the low quality of extracted RNA due to contaminants from the residue of phenol and guanidinium thiocyanate from the reagents. This study aimed to evaluate the improvement in the quality and concentration of RNA after the optimisation treatment. One-month-old tomato (Solanum lycopersicum) stem was used in this research. TRIzol or acid guanidinium thiocyanate-phenol-chloroform-based method was given optimisation treatments of the initial sample amount, twice chloroform extraction, overnight precipitation at low temperature, and three times final washing with ethanol. The results showed no significant improvement (p > 0.05) in the purity ratio A260/A280. At the same time, there was a significant improvement (p < 0.05) in RNA yield and purity ratio A260/A230. The quality of RNA was verified using agarose-formaldehyde electrophoresis gel. Eight of nine samples (89%) from the optimised group had better RNA integrity characterised by sharp bands for 28S and 18S rRNA. Furthermore, a representative sample from the optimised group was successfully synthesised into complementary DNA by reverse transcriptase-polymerase chain reaction (RT-PCR) with primers of the ubiquitin (UBI3) gene. To sum up, optimised TRIzol-based protocol provides meaningful insight to produce RNA with better quality and suitability for downstream applications.

  • Baskins, S., Bond, J., & Minor, T. (2019). Unpacking the growth in per capita availability of fresh market tomatoes.

  • Behnam, B., Bohorquez-Chaux, A., Castaneda-Mendez, O. F., Tsuji, H., Ishitani, M., & Becerra Lopez-Lavalle, L. A. (2019). An optimized isolation protocol yields high-quality RNA from cassava tissues (Manihot esculenta Crantz). FEBS Open Bio, 9(4), 814-825.

  • Bilgin, D. D., DeLucia, E. H., & Clough, S. J. (2009). A robust plant RNA isolation method suitable for Affymetrix GeneChip analysis and quantitative real-time RT-PCR. Nature Protocols, 4(3), 333–340.

  • Deepa, K., Sheeja, T. E., Santhi, R., Sasikumar, B., Cyriac, A., Deepesh, P. V., & Prasath, D. (2014). A simple and efficient protocol for isolation of high quality functional RNA from different tissues of turmeric (Curcuma longa L.). Physiology and Molecular Biology of Plants, 20(2), 263-271.

  • Farrell Jr., R. E. (2017). RNA methodologies: Laboratory guide for isolation and characterization (5th ed.). Academic Press.

  • Fengel, D., & Wegener, G. (1984). Wood - Chemistry, ultrastructure, reactions. Walter de Gruyter.

  • Ferniah, S. R., Kasiamdari, R. S., Priyatmojo, A., & Daryono, B. S. (2015). Expression of class II chitinase Gene in chilli (Capsicum annuum L.) as response to Fusarium oxysporum pathogen attack. Asian Journal of Plant Pathology, 9(3), 142-147.

  • Gallagher, S. R. (2017). Quantitation of DNA and RNA with absorption and fluorescence spectroscopy. Current Protocols in Immunology, 116(1), A.3L.1-A.3L.14.

  • Ghawana, S., Paul, A., Kumar, H., Kumar, A., Singh, H., Bhardwaj, P. K., Rani, A., Singh, R. S., Raizada, J., Singh, K., & Kumar, S. (2011). An RNA isolation system for plant tissues rich in secondary metabolites. BMC Research Notes, 4(1), 85.

  • Giovannoni, J. J. (2004). Genetic regulation of fruit development and ripening. The Plant Cell, 16(suppl. 1), S170–S180.

  • Hyakumachi, M., Nishimura, M., Arakawa, T., Asano, S., Yoshida, S., Tsushima, S., & Takahashi, H. (2012). Bacillus thuringiensis suppresses bacterial wilt disease caused by Ralstonia solanacearum with systemic induction of defense-related gene expression in tomato. Microbes and Environments, 28(1), 128-134.

  • Ishihara, T., Mitsuhara, I., Takahashi, H. & Nakaho, K. (2012). Transcriptome analysis of quantitative resistance-specific response upon Ralstonia solanacearum infection in tomato. PLOS One, 7(10), e46763.

  • Jaiswal, A. K., Alkan, N., Elad, Y., Sela, N., Philosoph, A. M., Graber, E. R., & Frenkel, O. (2020). Molecular insights into biochar-mediated plant growth promotion and systemic resistance in tomato against Fusarium crown and root rot disease. Scientific Reports, 10(1), 13934.

  • Li, Y., Chen, S., Liu, N., Ma, L., Wang, T., Veedu, R. N., & Jing, X. (2020). A systematic investigation of key factors of nucleic acid precipitation toward optimized DNA/RNA isolation. BioTechniques, 68(4), 191-199.

  • Liedl, B. E., Labate, J. A., Stommel, J. R., Slade, A., & Kole, C. (2013). Genetics, genomics, and breeding of tomato. CRC Press.

  • Liu, L., Han, R., Yu, N., Zhang, W., Xing, L., Xie, D., & Peng, D. (2018). A method for extracting high-quality total RNA from plant rich in polysaccharides and polyphenols using Dendrobium huoshanense. PLOS One, 13(5), e0196592.

  • Løvdal, T., & Lillo, C. (2009). Reference gene selection for quantitative real-time PCR normalization in tomato subjected to nitrogen, cold, and light stress. Analytical Biochemistry, 387(2), 238-242.

  • Milling, A., Babujee, L., & Allen, C. (2011). Ralstonia solanacearum extracellular polysaccharide is a specific elicitor of defense responses in wilt-resistant tomato plants. PLOS One, 6(1), e15853.

  • Rajakani, R., Narnoliya, L., Sangwan, N. S., Sangwan, R. S., & Gupta, V. (2013). Activated charcoal-mediated RNA extraction method for Azadirachta indica and plants highly rich in polyphenolics, polysaccharides and other complex secondary compounds. BMC Research Notes, 6(1), 125.

  • Rio, D. C. (2015). Denaturation and electrophoresis of RNA with formaldehyde. Cold Spring Harbor Protocols, 2015(2), 219-222.

  • Roy, D., Tomo, S., Modi, A., Purohit, P., & Sharma, P. (2020). Optimising total RNA quality and quantity by phenol-chloroform extraction method from human visceral adipose tissue: A standardisation study. MethodsX, 7, 101113.

  • Sambrook, J., & Russell, D. W. (2001). Molecular cloning: A laboratory manual (3rd ed.). Cold Spring Harbor Lab Press.

  • Toni, L. S., Garcia, A. M., Jeffrey, D. A., Jiang, X., Stauffer, B. L., Miyamoto, S. D., & Sucharov, C. C. (2018). Optimization of phenol-chloroform RNA extraction. MethodsX, 5, 599-608.

  • Uner, B., Kombeci, K., & Akgul, M. (2016). The utilization of tomato stalk in fiber production: NaOH and CaO pulping process. Wood Research, 61(6), 927-936.

  • Vasanthaiah, H. K., Katam, R., & 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(3), 42-51.

  • Vennapusa, A. R., Somayanda, I. M., Doherty, C. J., & Jagadish, S. K. (2020). A universal method for high-quality RNA extraction from plant tissues rich in starch, proteins and fiber. Scientific Reports, 10(1), 16887.

  • Wang, H. M., Yin, W. C., Wang, C. K., & To, K. Y. (2009). Isolation of functional RNA from different tissues of tomato suitable for developmental profiling by microarray analysis. Botanical Studies, 50, 115-125.

  • Yadeta, K., & Thomma, B. (2013). The xylem as battleground for plant hosts and vascular wilt pathogens. Frontiers in Plant Science, 4, 97.

  • Zhou, R., Yu, X., Zhao, T., Ottosen, C. O., Rosenqvist, E., & Wu, Z. (2019). Physiological analysis and transcriptome sequencing reveal the effects of combined cold and drought on tomato leaf. BMC Plant Biology, 19(1), 377.

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