Improved laser capture microdissection (LCM)-based method for isolation of RNA, including miRNA and expression analysis in woody apple bud meristem
- 157 Downloads
Isolation of high-quality RNA, including miRNA, from microscopic woody apple bud meristem using laser capture microdissection-based method.
It is often challenging to study the expression of microRNAs (miRNAs) or genes in less accessible inner tissues of tree species rich in polyphenols or polysaccharides. Here, we report a laser capture microdissection (LCM)-based method for efficient and cost-effective isolation and expression analysis of miRNAs and genes in the meristem tissue of woody apple bud. The tissue fixation, processing, infiltration, and sectioning steps were optimized for LCM-based excision and subsequent RNA isolation. Further, we have confirmed that RNA isolated from LCM-derived apple bud meristem contained miRNAs and was of good quantity and quality, sufficient for downstream expression analysis.
KeywordsBud meristem Gene expression Laser capture microdissection Malus MicroRNAs RNA isolation Small RNA Stem-loop RT-PCR Tree
Laser capture microdissection
SV acknowledges Department of Science and Technology-Science and Engineering Research Board (DST-SERB), Govt. of India, for National-Postdoctoral Fellowship (N-PDF file no. PDF/2016/002423). AKS and VG acknowledge funding from Department of Biotechnology (DBT), Govt. of India (research grant no. BT/PR12766/BPA/118/63/2015). We acknowledge LCM facility, other central instrument facility and internal grant of NIPGR. We thank Dr. Mohar Singh, ICAR-NBPGR, Regional Station Shimla, India for giving us access to collect apple bud samples. We thank the lab members (Shalini and Archita) for critical reading of the manuscript and valuable comments.
Compliance with ethical standards
Conflict of interest
The authors declare no competing interests.
- Abbott E, Hall D, Hamberger B, Bohlmann J (2010) Laser microdissection of conifer stem tissues: isolation and analysis of high quality RNA, terpene synthase enzyme activity and terpenoid metabolites from resin ducts and cambial zone tissue of white spruce (Picea glauca). BMC Plant Biol 10:106. https://doi.org/10.1186/1471-2229-10-106 CrossRefGoogle Scholar
- Nakazono M, Qiu F, Borsuk LA, Schnable PS (2003) Laser-capture microdissection, a tool for the global analysis of gene expression in specific plant cell types: identification of genes expressed differentially in epidermal cells or vascular tissues of maize. Plant Cell 15(3):583–596CrossRefGoogle Scholar
- Ohtsu K, Smith MB, Emrich SJ, Borsuk LA, Zhou R, Chen T, Zhang X, Timmermans MCP, Beck J, Buckner B, Janick-Buckner D, Nettleton D, Scanlon MJ, Schnable PS (2007) Global gene expression analysis of the shoot apical meristem of maize (Zea mays L.). Plant J 52(3):391–404. https://doi.org/10.1111/j.1365-313x.2007.03244.x CrossRefGoogle Scholar
- Roux B, Rodde N, Moreau S, Jardinaud MF, Gamas P (2018) Laser capture micro-dissection coupled to RNA sequencing: a powerful approach applied to the model legume Medicago truncatula in interaction with Sinorhizobium meliloti. In: Yamaguchi N (ed) Plant transcription factors. Methods in molecular biology, vol 1830. Humana Press, New York, pp 191–224CrossRefGoogle Scholar
- Sakai K, Taconnat L, Borrega N, Yansouni J, Brunaud V, Paysant-Le Roux C, Delannoy E, Magniette MLM, Lepiniec L, Faure JD, Balzergue S, Dubreucq B (2018) Combining laser-assisted microdissection (LAM) and RNA-seq allows to perform a comprehensive transcriptomic analysis of epidermal cells of Arabidopsis embryo. Plant Methods 14:78. https://doi.org/10.1186/s13007-018-0275-x CrossRefGoogle Scholar
- Takahashi H, Kamakura H, Sato Y, Shiono K, Abiko T, Tsutsumi N, Nagamura Y, Nishizawa NK, Nakazono M (2010) A method for obtaining high quality RNA from paraffin sections of plant tissues by laser microdissection. J Plant Res 123(6):807–813. https://doi.org/10.1007/s10265-010-0319-4 CrossRefGoogle Scholar