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Improved laser capture microdissection (LCM)-based method for isolation of RNA, including miRNA and expression analysis in woody apple bud meristem

  • Swati Verma
  • Vibhav Gautam
  • Ananda K. SarkarEmail author
Short Communication
  • 78 Downloads

Abstract

Main conclusion

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.

Keywords

Bud meristem Gene expression Laser capture microdissection Malus MicroRNAs RNA isolation Small RNA Stem-loop RT-PCR Tree 

Abbreviations

DEPC

Diethyl pyrocarbonate

LCM

Laser capture microdissection

TBA

Tert-butyl alcohol

Notes

Acknowledgements

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.

Supplementary material

425_2019_3127_MOESM1_ESM.pptx (2 mb)
Fig. S1 LCM system settings for laser-based excision of meristem tissue from apple bud sections (PPTX 2028 kb)
425_2019_3127_MOESM2_ESM.xlsx (16 kb)
Supplementary material 2 (XLSX 15 kb)

References

  1. 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
  2. Balestrini R, Gomez-Ariza J, Klink VP, Bonfante P (2009) Application of laser microdissection to plant pathogenic and symbiotic interactions. J Plant Interact 4:81–92CrossRefGoogle Scholar
  3. Blokhina O, Valerio C, Sokołowska K, Zhao L, Kärkönen A, Niittylä T, Fagerstedt K (2016) Laser capture microdissection protocol for xylem tissues of woody plants. Front Plant Sci 7:1965.  https://doi.org/10.3389/fpls.2016.01965 Google Scholar
  4. Daccord N, Celton JM, Linsmith G et al (2017) High-quality de novo assembly of the apple genome and methylome dynamics of early fruit development. Nat Genet 49:1099–1106CrossRefGoogle Scholar
  5. Espina V, Wulfkuhle JD, Calvert VS, VanMeter A, Zhou W, Coukos G, Geho DH, Petricoin EF 3rd, Liotta LA (2006) Laser-capture microdissection. Nat Protoc 1(2):586–603.  https://doi.org/10.1038/nprot.2006.85 CrossRefGoogle Scholar
  6. Gasic K, Hernandez A, Korban SS (2004) RNA extraction form different apple tissues rich in polyphenols and polysaccharides for cDNA library construction. Plant Mol Biol Rep 22:437a–437gCrossRefGoogle Scholar
  7. Gautam V, Sarkar AK (2015) Laser assisted microdissection, an efficient technique to understand tissue specific gene expression patterns and functional genomics in plants. Mol Biotechnol 57:299–308CrossRefGoogle Scholar
  8. Gautam V, Singh A, Singh S, Sarkar AK (2016) An efficient LCM-based method for tissue specific expression analysis of genes and miRNAs. Sci Rep 6:21577.  https://doi.org/10.1038/srep21577 CrossRefGoogle Scholar
  9. Gautam V, Singh A, Verma S, Kumar A, Kumar P, Mahima Singh S, Mishra V, Sarkar AK (2017) Role of miRNAs in root development of model plant Arabidopsis thaliana. Indian J Plant Phys 22(4):382–392.  https://doi.org/10.1007/s40502-017-0334-8 CrossRefGoogle Scholar
  10. Jyske TM, Suuronen JP, Pranovich AV, Laakso T, Watanabe U, Kuroda K, Abe H (2015) Seasonal variation in formation, structure, and chemical properties of phloem in Picea abies as studied by novel microtechniques. Planta 242(3):613–629.  https://doi.org/10.1007/s00425-015-2347-8 CrossRefGoogle Scholar
  11. Kerk NM, Ceserani T, Tausta SL, Sussex IM, Nelson TM (2003) Laser capture microdissection of cells from plant tissues. Plant Physiol 132(1):27–35CrossRefGoogle Scholar
  12. Kladnik A (2013) Maize kernels-fixation in FAA, embedding, sectioning and feulgen staining. Bioprotocol 3(15):e835.  https://doi.org/10.21769/BioProtoc.835 Google Scholar
  13. 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
  14. 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
  15. Porto DD, Bruneau M, Perini P, Anzanello R, Renou J-P, dos Santos HP, Fialho FB, Revers LF (2015) Transcription profiling of the chilling requirement for bud break in apples: a putative role for FLC-like genes. J Exp Bot 66(9):2659–2672CrossRefGoogle Scholar
  16. 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
  17. 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
  18. Scanlon MJ, Ohtsu K, Timmermans MCP, Schnable PS (2009) Laser microdissection-mediated isolation and in vitro transcriptional amplification of plant RNA. Curr Protoc Mol Biol 87(1):25A.3.1–25A.3.15.  https://doi.org/10.1002/0471142727.mb25a03s87 Google Scholar
  19. Singh A, Gautam V, Singh S, Sarkar Das S, Verma S, Mishra V, Mukherjee S, Sarkar AK (2018) Plant small RNAs: advancement in the understanding of biogenesis and role in plant development. Planta 248(3):545–558.  https://doi.org/10.1007/s00425-018-2927-5 CrossRefGoogle Scholar
  20. Sui X, Nie J, Li X, Scanlon MJ, Zhang C, Zheng Y, Ma S, Shan N, Fei Z, Turgeon R, Zhang Z (2018) Transcriptomic and functional analysis of cucumber (Cucumis sativus L.) fruit phloem during early development. Plant J 96(5):982–996CrossRefGoogle Scholar
  21. Takacs EM, Li J, Du C, Ponnala L, Janick-Buckner D, Yu J, Muehlbauer GJ, Schnable PS, Timmermans MCP, Sun Q, Nettleton D, Scanlon MJ (2012) Ontogeny of the maize shoot apical meristem. Plant Cell 24:3219–3234CrossRefGoogle Scholar
  22. 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
  23. Tucker MR, Okada T, Hu Y, Scholefield A, Taylor JM, Koltunow AMG (2012) Somatic small RNA pathways promote the mitotic events of megagametogenesis during female reproductive development in Arabidopsis. Development 139:1399–1404CrossRefGoogle Scholar
  24. Wu G, Park MY, Conway SR, Wang JW, Weigel D, Poethig RS (2009) The sequential action of miR156 and miR172 regulates developmental timing in Arabidopsis. Cell 138(4):750–759.  https://doi.org/10.1016/j.cell.2009.06.031 CrossRefGoogle Scholar
  25. Yuan TL, Huang WJ, He J, Zhang D, Tang WH (2018) Stage-specific gene profiling of germinal cells helps delineate the mitosis/meiosis transition. Plant Physiol 176(2):1610–1626.  https://doi.org/10.1104/pp.17.01483 CrossRefGoogle Scholar
  26. Zeng T, Holmer R, Hontelez J, Te Lintel-Hekkert B, Marufu L, de Zeeuw T, Wu F, Schijlen E, Bisseling T, Limpens E (2018) Host- and stage-dependent secretome of the arbuscular mycorrhizal fungus Rhizophagus irregularis. Plant J 94:411–425CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Swati Verma
    • 1
  • Vibhav Gautam
    • 1
    • 2
  • Ananda K. Sarkar
    • 1
    Email author
  1. 1.National Institute of Plant Genome ResearchNew DelhiIndia
  2. 2.Centre of Experimental Medicine and Surgery, Institute of Medical SciencesBanaras Hindu UniversityVaranasiIndia

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