几种提取白木香茎干总RNA方法的比较

Comparison of Several Methods for RNA Extraction from Stems of Aquilaria sinensis

目的:通过比较多种RNA提取方法,确定白木香茎干RNA提取的有效手段,为白木香伤害形成沉香药材的分子研究奠定基础。方法:取2年生白木香茎,分别用CTAB法、TRIzol法、异硫氰酸胍-SDS法、Qiagen试剂盒和Nor gen试剂盒法提取总RNA,通过琼脂糖凝胶电泳和紫外分光光度法测定其产量与质量,并通过cDNA合成及小样本克隆测序检查RNA的可用性。结果:异硫氰酸胍-SDS法及Norgen试剂盒法提出的RNA条带清晰,完整性好;D260nm/D280nm值可达2.0左右,纯度较高,其产量足够应用于cDNA合成;其他方法不能提出RNA。结论:异硫氰酸胍-SDS法及Norgen试剂盒法可用于白木香茎干RNA提取。

Objective： Aquilaria sinensis is one of the significant economic tree species for producing agarwood which is in high demand for medicine,incense and perfume across Asia,Middle East and Europe.As the agar wood is high-priced,the wild resource of A.sinensis was severely destroyed to obtain it.Agarwood is only formed in wounded or microbe infected stems,resulting in low production efficiency.To promote production efficiency and protect the resource of A.sinensis,regulation mechanisms of stress induced agarwood formation need to be studied.However,RNA extraction of A.sinensis stem is severely difficult because of the serious degradation.This work aimed at isolating high quality RNA from stems of A.sinensis for investigating the molecular mechanism of agar wood formation.Methods： We tested several methods（CTAB,TRIzol,guanidine isothiocyanate-SDS,Qiagen kit,and Norgen kit） for RNA extraction from stems of A.sinensis and evaluated of the extracted product through aga rose gel electrophoresis and absorbency analysis.The RNA was further tested by cDNA synthesis and sequencing.Results： The guanidine isothiocyanate-SDS method and Norgen kit could successfully isolate total RNA with high quality,agarose gel electrophoresis showed clear binds;D260nm/D280nm were about 2.0.The RNA yield was sufficient for cDNA synthesis.Other methods were failed to extract RNA.Conclusion： The guanidine isothiocyanate-SDS method and Norgen kit were suitable for RNA isolation from stems of A.sinensis.This is the first report of RNA isolation method from in vivo tissue of Aquilaria species.