Comparison of the methods of RNA isolation from Juglans regia L. buds

Abstract

In this paper, four RNA extraction methods (CTAB method, TRIzol method, D326A reagent and RP3301 Kit) for walnut buds were compared by agarose gel electrophoresis and spectrophotometry, and the optimal method was verified further by Reverse transcription and PCR amplification (RT-PCR). There was a certain degree of degradation for the extracted RNA by CTAB method, TRIzol method and D326A reagent. Moreover, protein was found in the CTAB method. For the three unsatisfactory methods, longer operation time could be the main reason that RNA could not be separated in time with polyphenols, polysaccharides and other secondary metabolites. RP3301 kit was suitable for the RNA extraction from walnut buds with high purity, simplicity, innocuity and no degradation. This research will provide a fundamental basis for the cloning and expression analysis of genes and other follow-up tests.

Keywords

Juglans regia L. / buds / total RNA / RP3301 kit

References

[1] Chen L, Zhu H, Li S, Lan X W, Huang Y Q, Wu Y S (2005). The contrast test of extracting total RNA from Panax pseudoginseng var. notoginseng. Letters in Biotechnology, 16(5): 528-530 (in Chinese)

[2] Fang G, Hammar S, Grumet R (1992). A quick and inexpensive method for removing polysaccharides from plant genomic DNA. Biotechniques, 13(1): 52-54, 56

[3] Fjellstrom R G, Parfitt D E, McGranahan G H (1994). Genetic relationships and characterization of Persian walnut (Juglans regia L.) cultivars using restriction fragment length polymorphisms (RFLPs). J Am Soc Hortic Sci, 119(4): 833-839

[4] John M E (1992). An efficient method for isolation of RNA and DNA from plants containing polyphenolics. Nucleic Acids Res, 20(9): 2381

[5] Li B G, Qi G H, Guo S P, Chen L Y (2008). A new early ripening, precocious and thin shell walnut cultivar-Lüzao. Acta Horticulturae Sinica, 35(7): 1088-1088 (in Chinese)

[6] Li J G, Guo S D (2004). Extraction of total RNA and cloning of conservative fragments of MADS-box gene by RT-PCR in upland cotton. Cotton Science, 16(1): 3-7 (in Chinese)

[7] Liu X J, Hong H B, Li M, Liu Y, Yang K Q (2008). An improved CTAB method for extracting total RNA from walnut. Shandong Agricultural Sciences, 1: 97-99 (in Chinese)

[8] Liu Z H, Zhao G H, Lu J S, Shui Y, Wu G (2008). Studies on content of cotton gossypol and characteristics of pest resistance. Xinjiang Agricultural Sciences, 45(3): 409-413 (in Chinese)

[9] Logemann J, Schell J, Willmitzer L (1987). Improved method for the isolation of RNA from plant tissues. Anal Biochem, 163(1): 16-20

[10] Nicese F P, Hormaza J L, Mcgranahan G H (1998). Molecular characterization and genetic relatedness among walnut (Juglans regia L.) genotype based on RAPD markers. Euphytica, 101(2): 199-206

[11] Potter D, Gao F Y, Aiello G (2002). Intersimple sequence repeat markers for fingerprinting and determining genetic relationships of walnut (Juglans regia L.) cultivars. J Am Soc Hortic Sci, 127(1): 75-81

[12] Wang G L, Fang H J (1998). Principles and Techniques of Plant Genetic Engineering. Beijing: Science Press, 611-614 (in Chinese)

[13] Wang H, Yan Y B, Zhang J P, Pei D (2009). Application of ITS sequence and SSR markers to study the relationship between Juglans regia and Juglans sigillata. Journal of Nanjing Forestry University (Natural Science Edition), 33(6): 35-39 (in Chinese)

[14] Wang H X, Zhao S G, Zhang Z H, Gao Y, Zhao Y P, Fang J, He F Q (2010). Genetic relationship and diversity of eight Juglans species in China estimated through AFLP analysis. Acta Horticulture 861, 143-150

[15] Wang Y, Wu G L, Li D K, Yang K, Hao B W (2003). Studies on the content of phenols in different parts of walnut tree. Journal of Fruit Science, 20(4): 325-327 (in Chinese)

[16] Woodhead M, Taylor M A, Davies H V, Brennan R M, McNicol R J (1997). Isolation of RNA from blackcurrant (Ribes nigrum L.) fruit. Mol Biotechnol, 7(1): 1-4

[17] Xi R T, Zhang Y P (1996). China Fruit Trees-Walnut. Beijing: China Forestry Publishing House (in Chinese)

[18] Xu F, Cai R, Chen L J, Chang J, Cheng S Y (2008). A method for isolation of high quality RNA from various Juglans regia tissues. Journal of Fruit Science, 25(2): 45-49 (in Chinese)

[19] Xu F, Li L L, Cheng H, Tang Y, Cheng S Y, Zhang W W (2010). Cloning and sequence analysis of a flavanone 3-Hydroxylase cDNA 3′end from Juglans regia. Acta Agriculturae Boreali-occidentalis Sinica, 19(1): 151-157 (in Chinese)

[20] Xu Y, Zhang M Y, An G N (2001). A new precocious walnut variety –Daifeng. Acta Horticulturae Sinica, 28(1): 87-87 (in Chinese)

[21] Yang K Q, Ma M, Sun C L, Sun M G, Guo Q R (2007). RAPD marker linked to precocious gene of walnut (Juglans regia L.) and its sequence analysis. Scientia Agricultura Sinica, 40(9): 2021-2027 (in Chinese)

[22] Yao J, Ma X L, Luan S, Wang Y, Sun L (2010). Determination of gossypol in cotton seed kernel in southern Xinjiang by high performance capillary electrophoresis. Journal of Xinjiang Medical University, 33(2): 119-123 (in Chinese)

[23] Zhang M Y, Shangguan L F, Xu Y, Xiang K, Fang J G (2010). Screening of RAPD primer for study of genetic relationship among walnut (Juglans regia L.) cultivars. Chinese Agricultural Science Bulletin, 26(2): 84-90 (in Chinese)

[24] Zhao J W, Li F Z, Cui T H, Zhang X (2009). Analysis of phenolic compounds in ‘Pingguoli’ pear cultivar during the growth period. Science and Technology of Food Industry, 3(6): 105-107 (in Chinese)

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