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Formamide‐based RNA Isolation at Above ZeroTemperatures from High Starch Cassava Tubers
Author(s) -
Bowrin Valerie,
RouseMiller Judy,
Sutton Fedora,
SirjuCharran Grace
Publication year - 2012
Publication title -
phytochemical analysis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.574
H-Index - 72
eISSN - 1099-1565
pISSN - 0958-0344
DOI - 10.1002/pca.2390
Subject(s) - formamide , chemistry , rna , rna extraction , starch , chromatography , biochemistry , organic chemistry , gene
ABSTRACT Introduction Cassava ( Manihot esculenta ) tubers are a main source of carbohydrate for a large percentage of people in the tropics. However, obtaining RNA from such high starch tubers proves problematic because gelation occurs during standard isolation procedures. In preliminary studies, formamide was used to homogenise tubers at room temperature and no gelation was observed. Objectives To develop a simple, reproducible formamide‐based procedure for RNA isolation from high starch tissues without the initial use of liquid nitrogen or lyophilisation. A second objective was to determine the impact of storage of formamide homogenates at 4°C on RNA integrity. Methodology Tubers were homogenised in 100% formamide at room temperature and stored for 1, 4 or 7 days at 4°C. Homogenates were centrifuged at 14000 × g for 10 min and RNA recovered from the supernatants by isopropanol precipitation followed by dissolution in guanidinium buffer. Protein was removed by standard phenol–chloroform–isoamyl alcohol extraction and RNA recovered by isopropanol precipitation. For immediate RNA isolation, formamide homogenates can be processed without incubation at 4°C. Results In formamide homogenates a non‐gelatinous white pellet was observed which was identified as starch by Lugol's staining. Intact RNA was observed by agarose gel electrophoresis. The RNA was successfully used in downstream RT‐PCR reactions. Conclusion Formamide maintained the integrity of the RNA during tissue processing at 25 ± 2°C and storage at 4°C. This room temperature formamide‐based RNA isolation procedure met the criteria of simplicity, low‐cost, reproducibility and addressed gelation problems associated with high starch tissues. Copyright © 2012 John Wiley & Sons, Ltd.