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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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.
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