Abstract. Recovering RNA of high quality and quantity is a prerequisite for ensuring representation of all expressed genes in a cDNA library. An efficient procedure for isolating RNA from bud, internodal shoot, flower, and fruit tissues of apple has been developed. This protocol does not involve the use of phenol, lyophilization, or ultracentrifugation. In addition, this protocol overcomes problems of both RNA degradation and low yield attributed to oxidation by polyphenolic compounds and coprecipitation with polysaccharides, both abundant components in apple fruit and flower tissues. 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 cDNA library construction, and it can be used for a number of downstream analyses, including Northern blot hybridization and reverse transcription-polymerase chain reaction (RT-PCR). With this modified protocol, 25-900 µg of total RNA is routinely obtained from 1 g of fresh material. This method is of low cost and easy to perform.
Full text†: This article, in detail, is available only in the electronic version of the Plant Molecular Biology Reporter.
Key words: apple, bud, cDNA libraries, flower, fruit, RNA extraction, shoot
Abbreviations: Chl:Iaa, chloroform-isoamylalcohol, CTAB, cetyltrimethylammonium bromide; DAP, days after pollination; DEPC, diethyl pyrocarbonate; EST, expressed sequence tag; FW, fresh weight; PVP, polyvinylpyrrolidone; RT-PCR, reverse transcription-polymerase chain reaction.
†Editor's note: Although the scientific content of this article has been reviewed, the full-text Web publication has not been edited in detail.