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Isolation of RNA from laser-capture-microdissected giant cells at early differentiation stages suitable for differential transcriptome analysis.

ABSTRACT: Plant organ gene expression profile analyses are complicated by the various cell types, and therefore transcription patterns, present in each organ. For example, each gall formed in roots following root knot nematode infection contains between four and eight specialized feeding cells (giant cells, GCs) embedded within hypertrophied root tissues. A recent goal in plant science has been the isolation of nematode feeding cell mRNAs for subsequent gene expression analysis. By adapting current protocols for different plant species and cells, we have developed a simple and rapid method for obtaining GCs from frozen tissue sections of tomato with good morphology and preserved RNA. The tissue sections obtained were suitable for the laser capture microdissection of GCs 6-7 days post-infection, and even of very early developing GCs (48-72 h post-infection), by fixation of tissue with ethanol-acetic acid, infiltration with sucrose and freezing in isopentane with optimal cutting temperature medium. This process was also successful for obtaining control vascular cells from uninfected roots for direct comparison with GCs. A minimum of about 300 GCs and 600 control vascular cells was required for efficient linear RNA amplification through in vitro transcription. Laser capture microdissection-derived RNA, after two rounds of amplification, was successfully used for microarray hybridization and validated with several differentially expressed genes by quantitative polymerase chain reaction. Consistent with our results, 117 homologous genes were found to be co-regulated in a previous microarray analysis of Arabidopsis galls at the same developmental stage. Therefore, we conclude that our method allows the isolation of a sufficient quantity of RNA with a high quality/integrity, appropriate for differential transcriptome analysis.

SUBMITTER: Portillo M

PROVIDER: S-EPMC6640446 | biostudies-literature | 2009 Jul

REPOSITORIES: biostudies-literature

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Isolation of RNA from laser-capture-microdissected giant cells at early differentiation stages suitable for differential transcriptome analysis.

Portillo Mary M Lindsey Keith K Casson Stuart S García-Casado Gloria G Solano Roberto R Fenoll Carmen C Escobar Carolina C

Molecular plant pathology 20090701 4

Plant organ gene expression profile analyses are complicated by the various cell types, and therefore transcription patterns, present in each organ. For example, each gall formed in roots following root knot nematode infection contains between four and eight specialized feeding cells (giant cells, GCs) embedded within hypertrophied root tissues. A recent goal in plant science has been the isolation of nematode feeding cell mRNAs for subsequent gene expression analysis. By adapting current protoc ...[more]

PMID: 19523105

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Project description:BACKGROUND:Formalin-fixed paraffin embedded (FFPE) tissue constitutes a vast treasury of samples for biomedical research. Thus far however, extraction of RNA from FFPE tissue has proved challenging due to chemical RNA-protein crosslinking and RNA fragmentation, both of which heavily impact on RNA quantity and quality for downstream analysis. With very small sample sizes, e.g. when performing Laser-capture microdissection (LCM) to isolate specific subpopulations of cells, recovery of sufficient RNA for analysis with reverse-transcription quantitative PCR (RT-qPCR) or next-generation sequencing (NGS) becomes very cumbersome and difficult. METHODS:We excised matched cancer-associated stroma (CAS) and normal stroma from clinical specimen of FFPE canine mammary tumours using LCM, and compared the commonly used protease-based RNA isolation procedure with an adapted novel technique that additionally incorporates a focused ultrasonication step. RESULTS:We successfully adapted a protocol that uses focused ultrasonication to isolate RNA from small amounts of deparaffinised, stained, clinical LCM samples. Using this approach, we found that total RNA yields could be increased by 8- to 12-fold compared to a commonly used protease-based extraction technique. Surprisingly, RNA extracted using this new approach was qualitatively at least equal if not superior compared to the old approach, as Cq values in RT-qPCR were on average 2.3-fold lower using the new method. Finally, we demonstrate that RNA extracted using the new method performs comparably in NGS as well. CONCLUSIONS:We present a successful isolation protocol for extraction of RNA from difficult and limiting FFPE tissue samples that enables successful analysis of small sections of clinically relevant specimen. The possibility to study gene expression signatures in specific small sections of archival FFPE tissue, which often entail large amounts of highly relevant clinical follow-up data, unlocks a new dimension of hitherto difficult-to-analyse samples which now become amenable for investigation.

Dataset Information

Isolation of RNA from laser-capture-microdissected giant cells at early differentiation stages suitable for differential transcriptome analysis.

Publications

Isolation of RNA from laser-capture-microdissected giant cells at early differentiation stages suitable for differential transcriptome analysis.

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