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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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Project description:Purpose: The goals of this study is to determine the best method of gene expression quantification (RNA-seq, Microarray, NanoString) and amplification kits adapted to low-input and/or low-quality RNA samples (FFPE samples) Methods: Mouse bladder cancer cell line (mouse bladder cancer cell line, BC57) and mouse normal mouse normal urothelium were fixed in formalin and embedded in paraffin (FFPE), andfesh frozen (FF) in liquid nitrogen. The total RNA of these 4 samples were tested in triplicate by 3 technologies (NanoString, RNA-seq and Microarray) and the results were compared to its reference (high-quality and high-input RNA of mouse bladder cancer cell line and mouse normal mouse normal urothelium). For RNA-sequencing, each sample was tested by two library contruction kits: SMARTer Stranded Total RNA-Seq Kit - Pico Input mammelian and Ovation SoLo NuGEN RNA-seq System with three input quantities: 50pg, 250pg and 2ng of total RNA, except for the SMARTer Stranded Total RNA-Seq Kit - Pico Input mammelian kit for which the minimum recommended quantity was 250pg of total RNA. RNA-seq based on poly(A) tail enrichment was considered as the reference and was done for the two FF samples at high amount (1µg of total RNA) . Results obtained with the two tested kits were compared to those based on poly(A) tail enrichment. To determine which is the best kit suitable for RNA-seq, low-input and low-quality RNA samples, we performed RNA-seq control quality metrics, principal component analysis, and a differential analysis between the mouse bladder cancer cell lines and the mouse normal mouse normal urothelium for each input quantity, library kit and method of sample preservation (FF or FFPE). Results: The Ovation SoLo NuGEN RNA-seq System library kit are recommended for quantification of gene expression of FFPE and FF samples at low-input (down to 50pg) whereas the SMARTer Stranded Total RNA-Seq Kit - Pico Input mammelian is only suitable for FF samples from 250pg of total RNA.

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

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