An improved total RNA isolation from secondary tissues of woody species for coding and non-coding gene expression analyses
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Pines, eucalypts and cork oak are ecologically and economically important species for wood, paper and cork industries. The identification of candidate genes and understanding of transcriptional regulation mechanisms related to wood and cork formation are considered as very important in breeding and conservation programmes for these species. The assessment of high-quality RNAs from different species, genotypes and tissues is required for transcriptomics studies. Most of the available protocols of total RNA isolation are suitable for needle or leaf tissue, including that reported by Chang et al. Here, an improved protocol for total RNA extraction from secondary tissues based on the protocol by Chang et al. is presented. The total RNA samples isolated from secondary tissues of Pinus sp., Eucalyptus globulus Labill. and Quercus suber L. showed good yield and quality. In the extracted RNA, the small RNA fraction was preserved, being suitable for different downstream approaches, such as expression analysis of coding and non-coding genes. It is considered that this protocol could be useful to other researchers who are working in the transcriptomics field of woody species.
KeywordsmiRNA Precursor Secondary Tissue Cork Plank Differentiate Xylem LiCl Precipitation Step
The work was partially supported by the projects funded by Fundação para a Ciência e a Tecnologia (FCT) co-financed by FEDER under the COMPETE-QREN Program (P-KBBE/AGR-GPL/0001/2010; PTDC/AGR-GPL/098179/2008; Sobreiro/0015/2009; PTDC/AGR-GPL/101785/2008; PEst-OE/EQB/LA0004/2011; PEst-OE/EQB/LA0023/2013; and PTDC/AGR-CFL/110988/2009) and the EU PROCOGEN Project No. 289841. JAPP thanks FCT by the Ciência 2008 programme research contract (FCT/POPH-QREN) and the SFRH/BPD/92207/2013 FCT’s Grant co-financed by FSE/POPH-QREN. AC and VC thank FCT for SFRH/BPD/68932/2010 and SFRH/BD/72982/2010 Grants, respectively, co-financed by FSE/POPH-QREN. CG and SP thank the research grants fellowships (BI) in the frame of P-KBBE/AGR-GPL/0001/2010 and PTDC/AGR-GPL/098179/2008; Projects. The authors thank I. Trindade for the miRNA Northern blot analysis; UTAD, Altri Florestal SA (Constância, Portugal) and Fundação João Lopes Fernandes for kindly providing the plant material.
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