کارآیی روش‌های استخراج RNA از برگ گیاه دارویی سرخارگل
محورهای موضوعی : بوم شناسی گیاهان زراعی
1 - بخش تحقیقات سلولی مولکولی، مدیریت بیوتکنولوژی کشاورزی منطقه مرکزی کشور- اصفهان، پژوهشکده بیوتکنولوژی کشاورزی ایران، سازمان تحقیقات،
2 - بخش تحقیقات ژنومیکس، مدیریت بیوتکنولوژی کشاورزی منطقه مرکزی کشور- اصفهان، پژوهشکده بیوتکنولوژی کشاورزی ایران، سازمان تحقیقات، آموزش و ترویج کشاورزی، تهران
کلید واژه: روش لیتیم کلراید, روش لیتیم کلراید/فنل, کیت RNAX-Plus, روش فنل/SDS,
چکیده مقاله :
سرخارگل گیاه دارویی ارزشمند حاوی سطوح بالایی از متابولیت های ثانویه می باشد که تا کنون روش مناسبی جهت استخراج RNA کل از آن معرفی نشده است. در پژوهش حاضر چهار روش استخراج RNA شامل روش لیتیم کلراید، کیت RNAX-Plus، فنل/SDS و لیتیم کلراید/فنل مورد بررسی قرار گرفت. به منظور تأیید کیفیت و خلوص RNA از روش اسپکتروفتومتری و الکتروفورز روی ژل آگارز 5/1% استفاده شد. نسبت بالای جذب محلول RNA در طول‌موج 260 نانومتر به طول موج 280 و 230 نانومتر، مشاهده RNA های ریبوزمی و عدم مشاهده اسمیر روی ژل به عنوان معیاری برای تعیین کیفیت نمونه‌ها در نظر گرفته شد. بالاترین کیفیت RNA حاوی 9/2 میکروگرم بر میکرولیتر در 100 میلی‌گرم برگ تازه در روش فنل/SDS و کمترین کیفیت RNA حاوی 2/1 میکروگرم در 100 میلی‌گرم برگ تازه در روش لیتیم کلراید/فنل مشاهده شد. بنابراین، روش فنل/SDS به عنوان مناسب ترین روش برای استخراج RNA برگ گیاه سرخارگل معرفی می گردد
Coneflower is a valuable medicinal plant containing high levels of secondary metabolites, but no suitable method for extraction of the entire RNA has been introduced yet. In present study, four methods of RNA extraction including lithium chloride, RNAX-Plus kit, phenol/SDS and lithium chloride/phenol were investigated. For confirming the quality and purity of RNA, spectrophotometer and agarose gel electrophoresis of 1.5% were used. The high absorption ratio of RNA solution at a wavelength of 260 nM to 280 and 230 nM, observation of ribosomal RNAs and the lack of smear on the gel were considered as criteria for determining samples quality. The highest and the lowest qualities of RNA contained 2.9 μg/100 mg of fresh leaves by phenol/SDS method and 1/2 μg/100 mg of fresh leaves by lithium chloride/phenol method, respectively. Therefore, phenol/SDS method is introducing as the most suitable method for RNA extraction from coneflower.
1. Bahador S, Rabiei B, Hasani- Kumleh SH (2014) Comparison of different methods for isolating of total RNA from leaf of three Thyme species rich in secondary metabolites. Iranian Journal of Rangelands and Forests Plant Breeding and Genetic Research 22(1): 11-24. [in Persian with English abstract]
2. Barrett B (2003) Medicinal properties of Echinacea: a critical review. Phytomedicine 10(1): 66-86.
3. Chaudhry B, Yasmin A, Husnain, T, Riazuddin S (1999) Mini-scale genomic DNA extraction from cotton. Plant Molecular Biology Reporter 17(3): 280-280.
4. Cheng YJ, Guo WW, Yi HL, Pang XM, Deng X (2003) An efficient protocol for genomic DNA extraction from Citrus species. Plant Molecular Biology Reporter 21(2): 177-178.
5. Chomczynski P, Sacchi N (1987) Single step method of RNA isolation by acid guanidinium thyocyanate–phenole-chlorophorm extraction. Analytical Biochemistry 162(1): 156-159.
6. Claros MG, Canovas FM (1998) Rapid high quality RNA preparation from pine seedlings. Plant Molecular Biology Reporter 16(1): 9-18.
7. Cruz RY, Laude RP, Diaz MGQ, Laurena AC, Mendioro MS, Mendoza EMT (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 L.). Philippine Agricultural Scientist 94(2): 118-123.
8. Deepa K, Sheeja TE, Santhi R, Sasikumar B, Cyriac A, Deepesh PV, 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.
9. Gonzalez-Mendoza D, Moreno AQ, Zapata-Perez O (2008) An improved method for the isolation of total RNA from Avicennia germinans leaves. Zeitschrift fur Naturforschung 63(1-2): 124-126.
10. Javdan-Asl M, Rajabi-Memari H, Nabati-Ahmadi D, Rahnama-Ghahfarokhi A (2016) Comparison of different genomic RNA extraction methods from the medicinal plant yarrow (Achillea millefolium). Plant Production (Scientific Journal of Agriculture) 39(2): 105-114. [in Persian with English abstract]
11. Kansal R, Kuhar K, Verma I, Niwas Gupta R, Kumar Gupta V, Koundal KR (2008) Improved and convenient method of RNA isolation from polyphenols and polysaccharide rich plant tissues. Indian Journal of Experimental Biology 46(12): 842-845.
12. Kolosova N, Miller B, Ralph S, Ellis BE, Douglas C, Ritland K, Bohlmann J (2004) Isolation of high-quality RNA from gymnosperm and Angiosperm trees. BioTechniques 36(5): 821-824.
13. MacRae EA (2007) Extraction of plant RNA. Methods in Molecular Biology 353: 15-24.
14. Omidbaigi, R. (2005). Production and processing of medicinal plants, Volume 2, Astan Quds Razavi Publication, 438 pages. Mashad, Iran. (in Farsi)
15. Rio DC, Ares M, Hannon GJ, Nilsen TW (2010) Purification of RNA by SDS solubilization and phenol extraction. Cold Spring Harbor Protoco 2010(6): 5438.
16. Rodrigues SM, Soares VL, Oliveira TM, Gesteira AS, Otoni WC, Costa MG (2007) Isolation and purification of RNA from tissues rich in polyphenols, polysaccharides, and pigments of annatto (Bixa orellana L.). Molecular Biotechnology 37(3): 220-224.
17. Rosas-Cardenas FF, Duran-Figueroa N, Vielle-Calzada JP, Cruz-Hernandez A, Marsch-Martinez N, de Folter S (2011) A simple and efficient method for isolating small RNAs from different plant species. Plant Methods 7(4): 1-7.
18. Rouholamin S, Zahedi B, Saei A, Nazarian Firouzabadi F (2015) Comparison of three genomic RNA extraction methods from pomegranate peel. New molecular cell biotechnology 5 (19): 39-45. [in Persian with English abstract]
19. Rubio–Pina JA, Zapata-Perez O (2011) Isolation of total RNA from tissues rich in polyphenols and polysaccharides of mangrove plants. Electronic Journal of Biotechnology 14(5): 1-8.
20. Tang Y, Simoneau AR, Xie J, Shahandeh B, Zi X (2008) Effects of the kava chalcone flavokawain A differ in bladder cancer cells with wild-type versus mutant p53. Cancer Prevention Research 1(6): 439-451.
21. Sambrook J, Russel DW (2001) Molecular Cloning: A Laboratory Manual. 3rd edition. Cold Spring Harbor Laboratory Press: New York.
22. Schneiderbauer A, Sandermann H, Ernst D (1991) Isolation of functional RNA from plant tissues rich in phenolic compounds. Analytical Biochemistry 197(1): 91-95.
23. Tousch D, Lajoix AD, Hosy E, Azay-Milhau J, Ferrare K, Jahannault C, Cros G, Petit P (2008) Chicoric acid, a new compound able to enhance insulin release and glucose uptake. Biochemical and Biophysical Research Communications 377(1): 131-135.
24. Wang L, Stegemann JP (2010) Extraction of high quality RNA from polysaccharide matrices using cetyltrim ethylammonium bromide. Biomaterials 31(7): 1612-1618.
25. Winfrey MR, Rott MA, Wortman AT (1997) Unraveling DNA: Molecular Biology for the Laboratory. Prentice-Hall, Inc: New York.
26. Zarei A, Zamani Z, Mousavi A, Fatahi R, Karimi Alavijeh M, Dehsara B, Salami SA (2012) An effective protocol for isolation of high-quality RNA from pomegranate seeds. The Asian and Australasian Journal of Plant Science and Biotechnology 6(1): 32-37.
