Efficient Isolation of High-quality Total RNA from Strawberry

in HortScience

Sufficient yields of high-quality RNA are needed for next-generation sequencing and high-throughput real-time polymerase chain reaction analyses. In the case of strawberry (Fragaria ×ananassa) fruits, successful RNA isolation requires removal of abundant inhibitory substances (polysaccharides and polyphenols) that greatly reduce quality and yield. In this study, we applied various combinations of RNA isolation protocols directed at reproductive organs. The best manual isolation method involved nonionic polymer and modified acid guanidinium thiocyanate-phenol-chloroform treatments followed by phenol/chloroform/isoamyl alcohol extraction. Compared with other methods, this approach gave significantly higher yields [84.0 µg/g fresh weight (FW)] of RNA of greater purity (A260/A280 = 1.99; A260/230 = 1.51). Better-quality RNA (A260/230 = 2.11) was obtained using an automated method, but the yield was lower (18.1 µg/g FW) than that obtained manually. This automated method consisted of pretreatment with nonionic polymer followed by a silica-based system extraction. Although RNA of sufficient quality [RNA Integrity Number (RIN) ≥ 6.5 and 28S/18S ≥ 1.0] for RNA sequencing was obtained from receptacles using both automated and manual methods, the manual method yielded high-quality RNA from achenes and anthers. The automatic method features 6-fold faster high-throughput capacity, whereas the manual method has wider applicability to different tissues.

Contributor Notes

We thank Drs. Takeshi Kurokura (Utsunomiya University), Yasuo Suzuki (Meijo University), Miho Iduhara (Biostir, Inc.), Takashi Kaneda (Promega), and Michio Kanechi (Kobe University) for helpful advice about RNA isolation. We also thank Hiroki Yoshikawa and Shunji Ohkochi (Kobe University) for their technical assistance. This work was supported by the Hyogo Alliance of Universities and Colleges for Innovation, Japan, the Japan Society for the Promotion of Science KAKENHI (grant numbers JP24658030 and JP18J10814), and the Sasakawa Scientific Research Grant from the Japan Science Society (grant number 29-429).

Corresponding author. E-mail: yuno@kobe-u.ac.jp.

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    Electropherograms of RNA isolated from different organs of strawberry. RNA was extracted from receptacles (upper) and achenes (middle) of ripe fruits and anthers (lower) of flowers in full bloom using manual (left) or automated (right) methods. The manual method included Fruit-mate and Sepasol treatments followed by phenol–chloroform–isoamyl alcohol extraction, and the automated method consisted of Fruit-mate pretreatment followed by application of the Promega Maxwell system (see also Table 2). Electropherograms were generated on an Agilent 2100 Bioanalyzer.The x- and y- axes represent nucleotides (nt) and relative fluorescence units (FU), respectively.

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    Analyses of gene transcript levels of Fra a 1 in strawberry by real-time polymerase chain reaction. RNA was extracted from receptacles of ripe fruits using manual or automated methods (see legend to Table 1). Relative transcript levels of Fra a 1.01 (A) and Fra a 1.02 (B) normalized against that of the elongation factor 1α gene (EF1α). Significant differences were not detected between RNA isolation methods as indicated by n.s. (P < 0.05; Student’s t test, n = 4).

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    Melting peak analyses of Fra a 1.01 (A) and Fra a 1.02 (B) conducted after polymerase chain reaction amplifications. RNA was extracted from receptacles of ripe fruits using manual (gray line) or automated (black solid line) methods (see also Fig. 2).

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