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.
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).
Amil-RuizF.Garrido-GalaJ.Blanco-PortalesR.FoltaK.M.Muñoz-BlancoJ.CaballeroJ.L.2013Identification and validation of reference genes for transcript normalization in strawberry (Fragaria × ananassa) defense responsesPLoS One8e70603
Amil-Ruiz,F.Garrido-Gala,J.Blanco-Portales,R.Folta,K.M.Muñoz-Blanco,J.Caballero,J.L.2013Identification and validation of reference genes for transcript normalization in strawberry (Fragaria × ananassa) defense responses8e70603)| false
ChenY.ChenY.ShiC.HuangZ.ZhangY.LiS.LiY.YeJ.YuC.LiZ.ZhangX.WangJ.YangH.FangL.ChenQ.2018SOAPnuke: A MapReduce acceleration-supported software for integrated quality control and preprocessing of high-throughput sequencing dataGigascience716
Chen,Y.Chen,Y.Shi,C.Huang,Z.Zhang,Y.Li,S.Li,Y.Ye,J.Yu,C.Li,Z.Zhang,X.Wang,J.Yang,H.Fang,L.Chen,Q.2018SOAPnuke: A MapReduce acceleration-supported software for integrated quality control and preprocessing of high-throughput sequencing data716)| false
ChristouA.GeorgiadouE.C.FilippouP.ManganarisG.A.FotopoulosV.2014Establishment of a rapid, inexpensive protocol for extraction of high quality RNA from small amounts of strawberry plant tissues and other recalcitrant fruit cropsGene537169173
Christou,A.Georgiadou,E.C.Filippou,P.Manganaris,G.A.Fotopoulos,V.2014Establishment of a rapid, inexpensive protocol for extraction of high quality RNA from small amounts of strawberry plant tissues and other recalcitrant fruit crops537169173)| false
HirakawaH.ShirasawaK.KosugiS.TashiroK.NakayamaS.YamadaM.KoharaM.WatanabeA.KishidaY.FujishiroT.TsuruokaH.MinamiC.SasamotoS.KatoM.NanriK.KomakiA.YanagiT.GuoxinQ.MaedaF.IshikawaM.KuharaS.SatoS.TabataS.IsobeS.N.2014Dissection of the octoploid strawberry genome by deep sequencing of the genomes of Fragaria speciesDNA Res.21169181
Hirakawa,H.Shirasawa,K.Kosugi,S.Tashiro,K.Nakayama,S.Yamada,M.Kohara,M.Watanabe,A.Kishida,Y.Fujishiro,T.Tsuruoka,H.Minami,C.Sasamoto,S.Kato,M.Nanri,K.Komaki,A.Yanagi,T.Guoxin,Q.Maeda,F.Ishikawa,M.Kuhara,S.Sato,S.Tabata,S.Isobe,S.N.2014Dissection of the octoploid strawberry genome by deep sequencing of the genomes of Fragaria species21169181)| false
Sánchez-SevillaJ.F.Cruz-RusE.ValpuestaV.BotellaM.A.AmayaI.2014Deciphering gamma-decalactone biosynthesis in strawberry fruit using a combination of genetic mapping, RNA-Seq and eQTL analysesBMC Genomics15218232
Sánchez-Sevilla,J.F.Cruz-Rus,E.Valpuesta,V.Botella,M.A.Amaya,I.2014Deciphering gamma-decalactone biosynthesis in strawberry fruit using a combination of genetic mapping, RNA-Seq and eQTL analyses15218232)| false
Sánchez-SevillaJ.F.VallarinoJ.G.OsorioS.BombarelyA.PoséD.MerchanteC.BotellaM.A.AmayaI.ValpuestaV.2017Gene expression atlas of fruit ripening and transcriptome assembly from RNA-seq data in octoploid strawberry (Fragaria × ananassa)Sci. Rep.713737
Sánchez-Sevilla,J.F.Vallarino,J.G.Osorio,S.Bombarely,A.Posé,D.Merchante,C.Botella,M.A.Amaya,I.Valpuesta,V.2017Gene expression atlas of fruit ripening and transcriptome assembly from RNA-seq data in octoploid strawberry (Fragaria × ananassa)713737)| false
SeveroJ.de OliveiraI.R.TiecherA.ChavesF.C.RombaldiC.V.2015Postharvest UV-C treatment increases bioactive, ester volatile compounds and a putative allergenic protein in strawberryLebensm. Wiss. Technol.64685692
Severo,J.de Oliveira,I.R.Tiecher,A.Chaves,F.C.Rombaldi,C.V.2015Postharvest UV-C treatment increases bioactive, ester volatile compounds and a putative allergenic protein in strawberry64685692)| false