一种适用于RT-PCR的石榴籽粒总RNA提取方法

An improved method for extracting total RNA used to RT-PCR from seeds of pomegranate

为从石榴籽粒中提取高质量的RNA,以便进行后续分子生物学研究,针对石榴籽粒富含次生代谢物的特点,采用CTAB法并进行了改良,利用氯仿/异戊醇替代其他方法中的"异硫氰酸胍"和"Trizol试剂"进行反复抽提去除蛋白,无水乙醇去除多糖,LiCl消化DNA。结果显示:改良CTAB法提取的石榴籽粒总RNA的28S rRNA、18SrRNA条带清晰,完整性较好;OD260 nm/OD280 nm比值为1.9960,纯度较高。在此基础上通过反转录、PCR扩增出符合预期大小的Actin基因片段,表明该方法提取的RNA可满足后续RT-PCR等分子生物学试验研究。

To extract high quality RNA from seeds of pomegranate for the molecular biology study,the modified CTAB method was used and improved according to the character of polyphenol secondary metabolites were rich in seeds of pomegranate.Guanidine isothiocyanate and Trizol reagent was replaced by chloroform/isoamyl alcohol to remove protein,and anhydrous ethanol was used to remove polysaccharide,LiCl to digest DNA.The results indicated that the RNA obtained from seeds of pomegranate with modified CTAB method had good integrity,the bands of 28S rRNA and 18S rRNA were clear,the OD260/OD280 was 1.9960.On this basis,the Actin gene fragment was expectedly and successfully obtained by reverse transcription and PCR amplification indicaed that the extracted RNA could be used for the following molecular biology operations.