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

Abstract

Me thods 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.

Low Temperature Increases Ethylene Sensitivity in Actinidia chinensis ‘Rainbow Red’ Kiwifruit

“…Total RNA was extracted from the frozen samples using the method for polysaccharide-rich fruit tissues by Ikoma et al (1996), and treated with DNase (RNeasy; QIAGEN, Hilden, Germany) to eliminate any DNA contamination. First-strand cDNA was synthesized from total RNA using a reverse transcription kit (TaqMan Reverse Transcription Reagents; Applied Biosystems Japan Ltd., Tokyo, Japan) according to the manufacturer's instructions, and was used as a template for amplifying cDNA fragments encoding the five ripening-related genes 1-aminocyclopropane-1-carboxylate (ACC) synthase gene ACS1, ACC oxidase gene ACO3, EIN-like gene EIL4, ethylene response gene ERF14, and polygalacturonase gene PGB.…”

Structure and Expression Levels of Alleles of Citrus Zeaxanthin Epoxidase Genes

“…Total RNA was extracted as described by Ikoma et al (1996) and purified with an RNeasy Minikit (Qiagen) with on-column DNase digestion. First-strand cDNAs were synthesized from 5 μg total RNA by reverse transcriptase with Oligo-(dT) primers using a Ready-To-Go You-Primed First Strand Kit (GE Healthcare).…”

Expression Quantitative Trait Loci Analysis of Carotenoid Metabolism-related Genes in Citrus

“…Among the AG population, wide variations were observed in expression levels of PDS, ZDS, LCYb, ZEP, NCED2, and NCED3, some of which exceeded fruit (more than three fruits per individual) of the AG population by the method described by Ikoma et al (1996). An RNeasy Minikit (Qiagen, Hilden, Germany) with on-column DNase digestion purified the RNA samples.…”