نوع مقاله : علمی - پژوهشی
نویسندگان
1 دانشجوی کارشناسی ارشد زراعت و اصلاح نباتات، دانشگاه شهید چمران اهواز
2 استادیار، گروه زراعت و اصلاح نباتات، دانشکده کشاورزی دانشگاه شهید چمران اهواز
چکیده
در مطالعات مولکولی، استخراج RNA یک مرحله بسیار مهم به شمار میرود و نتایج کارهای بعدی مانند Real-TIME PCR و RACE PCR به کیفیت و کمیت RNA استخراج شده بستگی دارد. تعدادی روش استاندارداستخراج RNA کل از بافت گیاه که برای گیاهان عادی مؤثر هستند، در دسترس میباشد، اما استخراج RNA کل در گیاهان با سطوح بالای کربوهیدرات و متابولیتهای ثانویه مانند ترکیبات فنولی مشکل میباشد. بومادران از گیاهان دارویی و صنعتی ارزشمند در مراتع ایران است که حاوی سطوح بالایی ار متابولیتهای ثانویه میباشد ولی تاکنون روش مناسبی جهت استخراج RNA کل از آن معرفی نگردیده است. این پژوهش با هدف شناسایی بهترین روش از بین سه روش استخراج RNA کل شامل استخراج با بافر (-Plus)RNXTM، لیتیمکلراید و فنل-کلروفورم از بافتهای گل و برگ بومادران انجام گردید. نتایج نشان داد که از بین سه روش، RNA استخراج شده با روش فنل-کلروفورم دارای کمیت و کیفیت بالایی (با طول موج 280/260) در محدوده قابل قبول 2-8/1 میباشد. این موضوع به دلیل حذف پلیساکاریدها و متابولیتهای ثانویه بهویژه پلیفنلها از محتویات سلولی بافت گیاه موجب استخراج بهتر RNA میگردد. به همین دلیل این روش جهت استخراج RNA از این گیاه ارزشمند در مطالعات بعدی پیشنهاد میگردد.
کلیدواژهها
موضوعات
عنوان مقاله [English]
Comparison of Different Genomic RNA Extraction Methods From the Medicinal Plant Yarrow (Achillea millefolium L.)
نویسندگان [English]
- Maryam Javdan Asl 1
- Hamid Rajabi Memari 2
- Daryoosh Nabati Ahmadi 2
- Afrasiyab Rahnama 2
1
2
چکیده [English]
Background and Objectives
RNA extraction is considered a crucial step in the molecular studies and the results of subsequent work such as, PCR, Real-TIME PCR and RACE PCR depend on the quality and quantity of the extracted RNA. Some standard methods are available in total RNA extraction of plant tissue, which are effective for the ordinary plants. But it is difficult to extract the total RNA from the plants with high levels of carbohydrates and secondary metabolites such as phenolic compounds. Yarrow is one of the valuable medicinal and industrial species of the pasture of Iran that contains high levels of secondary metabolites. So far, there is no report on a suitable method for extraction of total RNA from it.
Material and Methods
Total RNA extraction was done through three methods including RNXTM(-Plus), Lithium Chloride and Phenol-Chloroform. Then the quality and quantity of extraction RNA was evaluated.
Results
The results showed that among the three methods, extracted RNA with phenol-chloroform had high quantity and quality as the A 260/280 ratio was well within the accepted range of 1.8-2.
Discussion
Using phenol-chloroform method tends to remove polyphenols and polysaccharides from plant tissue cell contents and causes more efficient RNA extraction. Given of this, the phenol-chloroform method is recommended for total RNA extraction of valuable plant Yarrow in future studies.
کلیدواژهها [English]
- Yarrow
- RNA extraction
- Phenol-Chloroform
- Lithium Chloride
- RNXTM
- (-Plus) buffer
- Ahangaran, A., Mosahebi Mohammadi, G., Koohi Habibi, M., Khezri1, S., and Shahraeen, N. 2009. Use of rapid serological and nucleic acid-based methods for detecting the soybean mosaic virus. Journal of Agricultural Science and Technology, 11: 91-97.
- Benedek, B. and Kopp, B. 2007. Achillea millefolium revisited: Recent findings confirm the traditional use. Wien Med Wochenschr, 157(13-14): 312-314.
- Chang, S., Puryear, J., and Cairney, J. 1993. A simple and efficient method for isolating RNA from pine trees. Plant Molecular Biology Reporter, 11(2): 113-116.
- Channuntapipat, C., Sedgley, M., and Collins, G. 2001. Sequences of the cDNAs and genomic DNAs encoding the S1, S7, S8, and Sf alleles from almond, Prunus dulcis. Theoretical and Applied Genetics, 103(6-7): 1115-1122.
- Cruz, R.Y., Laude, R.P., Diaz, M.G., Laurena, A.C., Mendioro, M.S., and Mendoza, E.M. 2011. Gene for actin is a suitable internal reference for relative rt-pcr based expression analysis in normal and mutant makapuno endosperms of coconut (Cocos nucifera). Philippine Agricultural Scientist, 94(2): 118-123.
- Gonzalez-Mendoza, D., Quiroz Morenob, A., and Zapata-Perez, O. 2008. An improved method for the isolation of total RNA from Avicennia germinans Zeitschrift Fur Naturforschung, 63(3): 124-126.
- Hou, P., Xie, Z., Zhang, L., Song, Z., Mi, J., He, Y., and Li, Y. 2011. Comparison of three different methods for total RNA extraction from Fritillaria unibracteata: A rare Chinese medicinal plant. Journal of Medicinal Plants Research, 5(13), 2834-2838.
- Jones, C.S., Iannetta, P.P., Woodhead, M., Davies, H.V., McNicol, R.J., and Taylor, M. A. 1997. The isolation of RNA from raspberry (Rubus idaeus) fruit. Molecular Biotechnology, 8(3), 219-221.
- Lopez-Gomez, R. and Gomez-Lim, M.A. 1992. A method extracting intact RNA from fruits rich in polysaccharides using ripe mango mesocarp. Hort Science, 27(5): 440-442.
- MacRae, E. 2007. Extraction of plant RNA. Methods in Molecular Biology, 353: 15-24.
- Nasiri, N., Shokri, E., and Nematzadeh, G. 2011. Molecular cloning and characterization of vacuolar H+-ATPase subunit C gene in halophyte plant, Aeluropus Littoralis. New Cellular and Molecular Biotechnology, 2(5): 15-23. [In Farsi]
- Potrich, F.B., Allemand, A., da Silva, L.M., dos Santos, A.C., Baggio, C.H., Freitas, C.S., Mendes, D.A.G., Andre E., Werner, M.F., and Marques, M.C.A. 2010. Antiulcerogenic activity of hydroalcoholic extract of Achillea millefolium: Involvement of the antioxidant system. Journal of Ethno Pharmacology, 130(1): 85-92.
- Sharma, A.D., Gill, P.K., and Singh, P. 2002. DNA isolation from dry and fresh samples of polysaccharide-rich plants. Plant Molecular Biology Reporter, 20(4): 415-415.
- Tozyo, T., Yoshimura, Y., Sakurai, K., Uchida, N., Takeda, Y., Nakai, H., and Ishii, H. 1994. Novel antitumor sesquiterpenoids in Achillea millefolium. Chemical and Pharmaceutical Bulletin, 42(5): 1096-1100.
- Wadsworth, G.J., Redinbaugh, M.G., and Scandalios, J.G. 1988. A procedure for the small-scale isolation of plant RNA suitable for RNA blot analysis. Analytical Biochemistry, 172(1): 279-283.
- Wang, R.Y. and Ghabrial, S.A. 2002. Effect of aphid behavior on efficiency of transmission of soybean mosaic virus by the soybean-colonizing aphid, Aphis glycines. Plant Disease, 86(11): 1260-1264.
- Wang, T., Zhang, N., and Du, L. 2005. Isolation of RNA of high quality and yield from Ginkgo biloba Biotechnology Letters, 27(9): 629-633.
- Zargari, A. 1996. Medicinal plants. University of Tehran Press. 3: 106-113. [In Farsi]