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《Molecular Plant Breeding》 2013-02
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Different Methods for Extracting Total RNA and Their Application in Gene Cloning and Gene Expression Analysis in Soybean (Glycine max)

Guo Bin 1,2 Hou Siyu 1 Huang Kesheng 1,2 Lu Yang 1 Han Yuanhuai 1 Wang Yuguo 2* 1 Institute of Agricultural Bioengineerings, Taigu, 030801; 2 School of Agriculture, Shanxi Agricultural University, Taigu, 030801  
In order to find out the optimal method for extracting total RNA from leaves and flowers in soybean (Glycine max), extracting efficiency was compared among three methods of RNA extraction, RNApure plant kit, modified CTAB and hot borate. The yield and purity of total RNA were determined by nucleic acid and protein detector; integrity and quality of total RNA were analyzed by agrose gel electrophoresis, gene cloning and real-time PCR. The results showed that the yield of total RNA by using hot borate was six times that by the other methods (RNApure plant kit and modified CTAB). The value of OD260/OD280 was between 1.98 to 2.01; The electr-ophoresis result of RNA showed satisfactory bands with sharpness and integrity. ZAT9-like gene was obtained by cloning from the total RNA, sequenced to the nucleic acid sequence of 885 bp, showing up to 99% homology to ZAT9-like gene. In real-time PCR amplification, curve peak type and the melting curve peak type were better with RNA extracted with hot borate than the other methods. The quality of the RNA by hot borate was of high quality for gene cloning, real-time PCR and the other downstream application in molecular biology.
【Fund】: 山西省青年科技研究基金(2011021032-3)、山西省青年科技研究基金(2011021032-1);; 山西农业大学创新基金(2010028);; 山西省科技攻关项目(20120311005-3)共同资助
【CateGory Index】: S565.1
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