Submitted to: BMC Research Notes
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/20/2017
Publication Date: 6/29/2017
Citation: Honaas, L.A., Kahn, E.M. 2017. A practical examination of RNA isolation methods for European pear (Pyrus communis). BMC Research Notes. doi: 10.1186/s13104-017-2564-2. Interpretive Summary: Total RNA (all the RNA in a sample) contains messenger RNA, which can be quantified and then used to estimate gene activity (i.e. gene expression) in cells or tissues of interest. Reliable methods to extract RNA from various European pear fruit tissues are needed so that we can discover which genes are involved in, for instance, different riping patterns in the variety 'd'Anjou'. Tree fruit tissues are notoriously hard to extract, RNA isolation methods vary in their performance, and most have not been widely tested. In this paper we report a rigorous test on 'd'Anjou' pear fruit of several commercially available RNA isolation methods, plus a protocol we developed based on a published apple fruit protocol. We identified superior methods for reliably isolating RNA from pear fruit tissue, paving the way for powerful gene expression experiments in which all pear genes are monitored simultaneously via second generation sequencing.
Technical Abstract: 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 isolations methods, plus our modified version of a published cetyl trimethyl ammonium bromide (CTAB)-based method. 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, peel tissues were richer in RNA 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.