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Optimized Method for Robust Transcriptome Profiling of Minute Tissues Using Laser Capture Microdissection and Low-Input RNA-Seq

ABSTRACT: Purpose: To determine the optimal RNA extraction and library generation protocols for mouse hippocampal tissue acquired by laser capture microdissection (LCM). Methods: Hippocampal subregion CA2 was captured from eight micron fresh frozen brain sections from AMIGO2 EGFP mice. Total RNA was extracted using the PicoPure (LCM standard) and QIAGEN micro RNeasy RNA extraction kits. RNA quantity and quality was assessed using a Bioanalzyer. Resulting RNA was used to generate cDNA libraries using NuGEN or SMARTer low input RNA-Seq library kits. We compared the effects of RNA quality and library generation approach to determine the methods that detected the greatest number of genes/exons with even coverage using minimal rounds of PCR. Results: We determined that the QIAGEN RNA extraction kit resulted in far superior RNA compared to the Picopure RNA extraction kit. We also found the NuGEN library generation kit that depletes ribosomal RNA towards the end of the protocol, led to higher cDNA library yields that required fewer rounds of PCR while providing even gene coverage and detection.

ORGANISM(S): Mus musculus

PROVIDER: GSE95257 | GEO | 2017/06/02

SECONDARY ACCESSION(S): PRJNA376510

REPOSITORIES: GEO

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