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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

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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.

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Fig. 1
Fig. 2
Fig. 3

[flowchart is modified from our earlier report as per our current method (Gautam et al. 2016)]

Abbreviations

DEPC:

Diethyl pyrocarbonate

LCM:

Laser capture microdissection

TBA:

Tert-butyl alcohol

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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.

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  1. Vibhav Gautam

    Present address: Centre of Experimental Medicine and Surgery, Institute of Medical Sciences, Banaras Hindu University, Varanasi, 221005, India

Authors and Affiliations

  1. National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi, 110067, India

    Swati Verma, Vibhav Gautam & Ananda K. Sarkar

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Fig. S1

LCM system settings for laser-based excision of meristem tissue from apple bud sections (PPTX 2028 kb)

Supplementary material 2 (XLSX 15 kb)

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Verma, S., Gautam, V. & Sarkar, A.K. Improved laser capture microdissection (LCM)-based method for isolation of RNA, including miRNA and expression analysis in woody apple bud meristem. Planta 249, 2015–2020 (2019). https://doi.org/10.1007/s00425-019-03127-0

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