An efficient LCM-based method for tissue specific expression analysis of genes and miRNAs
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Title |
An efficient LCM-based method for tissue specific expression analysis of genes and miRNAs
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Creator |
Gautam, Vibhav
Singh, Archita Singh, Sharmila Sarkar, Ananda K. |
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Subject |
Gene expression analysis
Isolation, separation and purification |
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Description |
Accepted date: 26 January 2016
Laser Capture Microdissection (LCM) is a powerful tool to isolate and study gene expression pattern of desired and less accessible cells or tissues from a heterogeneous population. Existing LCM-based methods fail to obtain high quality RNA including small RNAs from small microdissected plant tissue and therefore, are not suitable for miRNA expression studies. Here, we describe an efficient and cost-effective method to obtain both high quality RNA and miRNAs from LCM-derived embryonic root apical meristematic tissue, which is difficult to access. We have significantly modified and improved the tissue fixation, processing, sectioning and RNA isolation steps and minimized the use of kits. Isolated RNA was checked for quality with bioanalyzer and used for gene expression studies. We have confirmed the presence of 19-24 nucleotide long mature miRNAs using modified stem-loop RT-PCR. This modified LCM-based method is suitable for tissue specific expression analysis of both genes and small RNAs (miRNAs). We acknowledge NIPGR internal grants and Ramalingaswami Fellowship (# BT/HRD/35/02/06/2008) to AKS from Department of Biotechnology (India).We are also thankful to the central instrumentation facility of NIPGR for providing necessary experimental set up. V.G., A.S. and S.S. thanks Council of Scientific and Industrial Research (CSIR, India) for fellowship. We thank laboratory members (Alka Singh and Ashutosh Kumar) for careful reading and comments. |
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Date |
2016-02-12T05:40:02Z
2016-02-12T05:40:02Z 2016 |
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Type |
Article
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Identifier |
Scientific Reports, 6: 21577
2045-2322 http://172.16.0.77:8080/jspui/handle/123456789/622 http://www.nature.com/articles/srep21577 10.1038/srep21577 |
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Language |
en_US
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Publisher |
Nature Publishing Group
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