Isolation and Evaluation of RNA from Polysaccharide-rich Tissues in Fruit for Quality by cDNA Library Construction and RT-PCR.

Abstract

Methods for extracting total RNA from various tissues of satsuma mandarin and kiwifruit were examined. Satisfactory yields in total RNA were obtained from tissues with the exception of the albedo tissue of satsuma mandarin, by using a modification of the conventional extraction method devised for fruit tissues by Lopez-Gomez and Gomez-Lim (1992). In the albedo tissue, the polysaccharides may interfere with RNA extraction but further modification of the extraction method improved its yield. The modification involved repeated back extraction, chloroform/isoamyl alcohol extraction, and increasing the volume of the aqueous phase before precipitating RNA with LiCl (3 M final concentration). From the total RNA, poly (A)+RNA was purified using an oligo (dT) -cellulose column. The poly (A)+RNA could be successfully used for Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and the construction of a cDNA library. This modified protocol is applicable to other fruit tissues rich in polysaccharides.