A method for Isolation of High Quality RNA from Various Tissues of Castanea mollissima

摘要

RNA extraction from Castanea mollissima tissues is difficult due to the presence of rich polysaccharides and polyphenolic compounds.In this study,an improved protocol was tried to isolate total RNA in high yield and integrity from Chestnut leaves,green fruit,shoot,flower and root,which contained high levels of polysaccharides and polyphenolic secondary metabolites.Polyvinylpolypyrrolidone at 2％ and β-mercaptoethanol at 3％ were added to the standard CTAB extraction buffer,and after chloroform and phenol extraction followed by ethanol/sodium acetate precipitation,second extraction was introduced to remove coprecipitated polysaccharides.Isolated RNA is of high quality and is undegraded as assessed by spectrophotometric readings and electrophoresis in denaturing agarose gels.RNA quality is further assessed following its use in reverse transcription and Northern blot hybridization,and it can be used for a number of downstream work,including RACE and cDNA library construction.