A practical examination of RNA isolation methods for European pear (Pyrus communis)

Abstract

Abstract Objective With the goal of identifying fast, reliable, and broadly applicable RNA isolation methods in European pear fruit for downstream transcriptome analysis, we evaluated several commercially available kit-based RNA isolation methods, plus our modified version of a published cetyl trimethyl ammonium bromide (CTAB)-based method. Results We confirmed previous work indicating chaotropic agent-based kits produced sufficient, high-quality RNA in freshly harvested, mature ‘Bartlett’ fruit. However, RNA isolation from ‘d’Anjou’ pear peel and especially cortical tissues of fruit stored for 11 months proved challenging to all but our modified CTAB-based method. Generally, more RNA was recovered from peel tissues than cortical tissues. Less toxic dithiothreitol was confirmed to be an acceptable reducing agent as its substitution for 2-mercaptoethanol often yielded high quality RNA. Finally, we present evidence that erroneous signals in the 5S region of Bioanalyzer RNA size plot histograms, that interfered with RNA integrity number calculation, were small RNA fragments that are reduced by simple cleanup procedures, not artifacts as previously reported.

Keywords

• RNA
• Pyrus communis
• 2-mercaptoethanol
• Dithiothreitol
• Cetyl-trimethyl-ammonium-bromide
• Bioanalyzer