Record Details

An efficient LCM-based method for tissue specific expression analysis of genes and miRNAs

NIPGR Digital Knowledge Repository (NDKR)

View Archive Info
 
 
Field Value
 
Title An efficient LCM-based method for tissue specific expression analysis of genes and miRNAs
 
Creator Gautam, Vibhav
Singh, Archita
Singh, Sharmila
Sarkar, Ananda K.
 
Subject Gene expression analysis
Isolation, separation and purification
 
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.
 
Date 2016-02-12T05:40:02Z
2016-02-12T05:40:02Z
2016
 
Type Article
 
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
 
Language en_US
 
Publisher Nature Publishing Group