PERBANDINGAN METODE EKSTRAKSI RIBO NUCLEIC ACID YANG BERBEDA PADA JARINGAN MANTEL KERANG BIRU (Mytilus edulis)
Abstract
Kerang biru (Mytilus edulis) merupakan salah satu sentinel spesies yang dapat bertahan hidup di berbagai kondisi lingkungan, bahkan di daerah dengan tekanan tinggi, namun kemampuan fisiologisnya masih belum banyak diketahui hingga saat ini. Regulasi fisiologis hewan dapat diketahui dengan mengetahui karakteristik genotip hewan melalui analisis genomik. Salah satu tahap yang diperlukan dalam analisis genomik adalah ekstraksi RNA. Perolehan kualitas dan kuantitas RNA yang baik merupakan langkah awal yang penting untuk analisis genomik selanjutnya. Penelitian ini bertujuan untuk membandingkan metode ekstraksi RNA yang berbeda pada jaringan mantel kerang biru agar dapat dihasilkan kualitas dan kuantitas RNA yang baik untuk analisis genomik. Dua metode ektraksi RNA yaitu menggunakan RNAqueous Phenol-free total RNA Isolation dan TRIzolTM. Reagent. Data berupa hasil deskriptif dan kuantitatif, RNA yang telah berhasil diekstraksi dinilai kualitas dan kuantitasnya dengan menggunakan alat Agilent 5300 Fragment Analyzer. Penggunaan RNAqueous Phenol-free total RNA Isolation dalam mengekstraksi jaringan mantel kerang biru tidak dapat dilakukan dengan baik. Penggunaan metode ekstraksi RNA dengan Kit TRIzolTM Reagent menghasilkan ekstrak RNA jaringan mantel kerang biru dengan nilai konsentrasi total RNA berkisar 48,91-392,38 ng mL-1, nilai RNA Quality Number (RQN) berkisar 7,6-9,6 dan rasio 28S:18S berkisar 0-3,5. Metode TRIzolTM Reagent kit memiliki efektifitas lebih baik dalam menghasilkan ekstrak RNA pada jaringan mantel kerang biru dengan kualitas dan kuantitas RNA yang baik.
The blue mussel (Mytilus edulis) is a sentinel species that can survive in various environmental conditions, even in high pressure areas, but its physiological abilities has not been widely known. Marine physiological regulation can be known by knowing the characteristics of the biota genotype through genomic analysis. One of the steps required in genomic analysis is RNA extraction. Obtaining good quality and quantity of RNA is an important first step for further genomic analysis. This study aimed to compare different RNA extraction methods in blue mussel mantle tissue, so that it is expected to produce good RNA for genomic analysis. Two RNA extraction methods used were RNAqueous Phenol-free total RNA Isolation and TRIzolTM. Reagents. The data were in the form of descriptive and quantitative results, the quality and quantity of RNA that has been successfully extracted is assessed using the Agilent 5300 Fragment Analyzer. The use of RNAqueous Phenol-free total RNA Isolation in extracting mantle tissue of blue mussel cannot be carried out well. The use of the RNA extraction method with the TRIzolTM Reagent Kit produced RNA extract of blue mussel mantle tissue with total RNA concentration values ranging from 48,91-392,38 ng mL-1, RNA quality number (RQN) ranging from 7.6-9.6 and ratio of 28S:18S ranging from 0-3.5. The TRIzolTM Reagent kit method had better effectiveness in producing RNA extract from blue mussel mantle tissue with good quality and quantity of RNA.
Keywords
Full Text:
PDFReferences
Adiputra, J., Hidayat, S. H., & Damayanti, T. A. (2013). Evaluasi tiga metode preparasi RNA total untuk deteksi Turnip mosaic potyvirus dari benih Brassica rappa dengan Reverse Transcription-Polymerase Chain Reaction. Jurnal Fitopatologi Indonesia, 8(2), 44-49. https://doi.org/10.14692/jfi.8.2.44
Anantharaman, S., & Craft, J. A. (2012). Annual variation in the levels of transcripts of sex-specific genes in the mantle of the common mussel, Mytilus edulis. PLOS ONE, 7(11), e508161. https://doi.org/10.1371/journal.pone.0050861
Aranda, R. IV., Dineen, S. M., Craig, R. L., Guerrieri, R. A., & Robertson, J. M. (2009). Comparison and evaluation of RNA quantification methods using viral, prokaryotic, and eukaryotic RNA over a 104 concentration range. Analytical Biochemistry, 387(1),122-127. https://doi.org/10.1016/j.ab.2009.01.003
Becker, C., Hammerle-Fickinger, A., Riedmaier, I., & Pfaffl, M. W. (2010). mRNA and microRNA quality control for RT-qPCR analysis. Methods, 50(4), 237-243. https://doi.org/10.1016/j.ymeth.2010.01.010
Fleige, S., Walf, V., Huch, S., Prgomet, C., Sehm, J., & Pfaffl, M. W. (2006) Comparison of relative mRNA quantification models and the impact of RNA integrity in quantitative real-time RT-PCR. Biotechnology Letters, 28, 1601-1613. https://doi.org/10.1007/s10529-006-9127-2
Food and Agriculture Organization of the United Nations. (2018). The state of world fisheries and aquaculture. Meeting the sustainable development goals. Food and Agriculture Organization of the United Nations.
Gayral, P., Weinert, L., Chiari, Y., Tsagkogeorga, G., Ballenghien, M., & Galtier, N. (2011). Next-generation sequencing of transcriptomes: A guide to RNA isolation in nonmodel animals. Molecular Ecology Resources, 11(4), 650-661. https://doi.org/10.1111/j.1755-0998.2011.03010.x
Gosling, E. (2022). Marine mussels: Ecology, physiology, genetics and culture. John Wiley & Sons Ltd.
Guillou, E., Cyr, C., Laplante, J.-F., Bourque, F., Toupoint, N., & Tremblay, R. (2020). Commercial performance of blue mussel (Mytilus edulis, L.) stocks at a microgeographic scale. Journal of Marine Science and Engineering, 8(6), 382. https://doi.org/10.3390/jmse8060382
Habib, S. H., Saud, H. M., & Kausar, H. (2014). Efficient oil palm total RNA extraction with a total RNA extraction kit. Genetics and Molecular Research, 13(2), 2359-2367. https://doi.org/10.4238/2014.April.3.8
Kemfack, A. M., Hernandez-Morato, I., Moayedi, Y., & Pitman, M. J. (2022). An optimized method for high-quality RNA extraction from distinctive intrinsic laryngeal muscles in the rat model. Scientific Reports, 12, 21665. https://doi.org/10.1038/s41598-022-25643-y
Kiewe, P., Gueller, S., Komor, M., Stroux, A., Theil, E., & Hoffman, W. K. (2009). Prediction of qualitative outcome of oligonucleotide microarray hybridization by measurement of RNA integrity using the 2100 BioanalyzerTM capillary electrophoresis system. Annals of Hematology, 88, 1177-1183. https://doi.org/10.1007/s00277-009-0751-5
Lesser, M. P., & MacManes, M. (2016). Transcriptomic resources for the rocky intertidal blue mussel Mytilus edulis from the Gulf of Maine. Journal of Shellfish Research, 35(2), 435–465. https://doi.org/10.2983/035.035.0218
Pereira, W. J., Bassinello, P. Z., Brondani, C., & Vianello, R. P. (2017). An improved method for RNA extraction from common bean seeds and validation of reference genes for qPCR. Crop Breeding and Applied Biotechnology, 17, 150-158. http://dx.doi.org/10.1590/1984-70332017v17n2a22
Pocernich, C., & Steve, S. (2019). Quality metrics for nucleic acids with the Agilent Fragment Analyzer and Femto Pulse Systems. Agilent Technologies, Inc.
Schroeder, A. S., Mueller, O., Stocker, S., Salowsky, R., Leiber, M., Gassmann, M., Ligthfoot, S., Menzel, W., Granzow, M., & Ragg, T. (2006). The RIN: an RNA integrity number for assigning integrity value to RNA measurements. BMC Molecular Biology, 7, 3. https://doi.org/10.1186/1471-2199-7-3
Seed, R. (1976). Ecology. In: B. L. Bayne (Ed.), Marine mussels: Their ecology and physiology (pp. 13–60). Cambridge University Press.
Simbolon, A. R. (2018). Analisis risiko kesehatan pencemaran timbal (Pb) pada kerang hijau (Perna viridis) di Perairan Cilincing Pesisir DKI Jakarta. Oseanologi dan Limnologi di Indonesia, 3(3), 197-208. https://doi.org/10.14203/oldi.2018.v3i3.207
Tan, K., & Conaco, C. (2021). Characterization of the hidden break in giant clam 28S ribosomal RNA. Journal of Molluscan Studies, 87(3), eyab029. https://doi.org/10.1093/mollus/eyab029
Tesena, P., Korchunjit, W., Taylor, J., Wongtawan, T. (2017). Comparison of commercial RNA extraction kits and qPCR master mixes for studying gene expression in small biopsy tissue samples from the equine gastric epithelium. Journal of Equine Science, 28(4), 135-141. https://doi.org/10.1294/jes.28.135
Walker, T. R., Macaskill, D., & Weaver, P. (2013). Blue mussels (Mytilus edulis) as bioindicators of stable water quality in Sydney Harbour during remediation of the Sydney Tar Ponds, Nova Scotia, Canada. Water Quality Research Journal, 48(4), 358-371. https://doi.org/10.2166/wqrjc.2013.014
Winnebeck, E. C., Millar, C. D., & Warman, G. R. (2010). Why does insect RNA look degraded?. Journal of Insect Science, 10(1), 159. https://doi.org/10.1673/031.010.14119
Wong, K.-S., & Pang, H.-M. (2013). Simplifying HT RNA quality and quantity analysis: Automated CE system designed to improve rapid assessment. Genetic Engineering & Biotechnology News, 33(2), 17. https://doi.org/10.1089/gen.33.2.09
Yarra, T., Gharbi, K., Blaxter, M., Peck, L. S., & Clark, M. S. (2016). Characterization of the mantle transcriptome in bivalves: Pecten maximus, Mytilus edulis and Crassostrea gigas. Marine Genomics, 27, 9–15. https://doi.org/10.1016/j.margen.2016.04.003
Zhang, H., Gaoyou, Yao., & Maoxian, He. (2022). Transcriptome analysis of gene expression profiling from the deep sea in situ to the laboratory for the cold seep mussel Gigantidas haimaensis. BMC Genomics, 23, 828. https://doi.org/10.1186/s12864-022-09064-9
Zulaeha, S., Purwoko, D., Cartealy, I., Tajuddin, T., Karyanti, Khairiyah, H. (2019). Perbandingan tiga kit ekstraksi RNA untuk analisis transkriptomika pada kelapa sawit (Elaeis guineensis Jacq.). Jurnal Bioteknologi & Biosains Indonesia, 6(1), 118-129. https://doi.org/10.29122/jbbi.v6i1.3372
DOI: http://dx.doi.org/10.15578/jra.18.2.2023.129-139
Jurnal Riset Akuakultur is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License.
