樟树不同组织总RNA提取方法比较及RT-PCR检测

采用Invitrogen Total RNA Purification Kit直接提取法,Trizol试剂盒法和本实验室改良的CTAB法,分别提取樟树不同组织(根、茎、叶和花)的总RNA,并对提取效果进行了检测和比较分析。结果表明:(1)采用本实验室改良的CTAB法提取的RNA完整性好,无杂质污染且得率高,稳定性好,可满足半定量RT-PCR等试验需要;而Invitrogen Total RNA Purification Kit直接提取法和Trizol试剂盒法提取的樟树不同组织总RNA,结果均不理想,存在RNA 28S rRNA谱带模糊不清、有DNA污染、降解严重和得率低等问题,这两种方法均不适用于樟树不同组织RNA的提取。(2)樟树不同组织总RNA提取的得率不同,其中叶片的总RNA得率最高,达到782.34 mg/kg;茎的得率最低,为514.33 mg/kg。(3)以Actin作为内参基因,对樟树根、茎、叶和花中的Fad2基因片段进行半定量RT-PCR检测,结果表明,Fad2基因片段在樟树叶片中的表达量相对较高,而在花中的表达量较低。

Comparative Isolation Methods for Total RNA from Different Tissues and Examination with RT-PCR in Cinnamomum camphora

The total RNA isolated from different tissues （root, stem, leaf and flower） in Cinnamomum camphora were extracted by Invitrogen Total RNA Purification Kit, common Trizol Kit and the improvement CTAB method, and the isolation effects of the three methods were tested and compared. The results showed as follow： （1） The improved CTAB could extract high-quality and high-integrity RNA, which could be used for semi-quantitative RT-PCR and so on. It was in an indistinct way of the total RNA from the different tissues in C. camphora by Invitrogen Total RNA Purification Kit extraction. RNA extracted was immingled by some DNA and was not enough for molecular biology experiment. Total RNA could not be extracted by Trizol method, the degradation of total RNA was seriously and the yield was very low. Trizol method and Invitrogen Total RNA Purification Kit were not suitable for RNA extraction from C. camphora. （2） The yield of RNA from leaves was 782.34 mg/kg, which higher than the yield from stems （514.33 mg/kg）. （3） RT - PCR was used to determine Fad2 gene expression with Actin gene as comparison. The results showed that the expression of Fad2 gene showed high level in leaf and low level in flower.