Isolation of RNA from laser-capture-microdissected giant cells at early differentiation stages suitable for differential transcriptome analysis.
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.
Portillo
Mary
M
Universidad de Castilla-La Mancha, Toledo, Spain.
Lindsey
Keith
K
Casson
Stuart
S
García-Casado
Gloria
G
Solano
Roberto
R
Fenoll
Carmen
C
Escobar
Carolina
C
D016616:Cryoultramicrotomy; D004586:Electrophoresis; D020869:Gene Expression Profiling / Q000379:methods*; D018506:Gene Expression Regulation, Plant; D015726:Giant Cells / Q000166:cytology / Q000378:metabolism*; D007834:Lasers; D018551:Solanum lycopersicum / Q000166:cytology / Q000235:genetics* / Q000254:growth & development*; D042282:Microdissection / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis; D016612:Paraffin Embedding; D018749:RNA, Plant / Q000302:isolation & purification*; D015203:Reproducibility of Results; D013696:Temperature; D016707:Tissue Fixation
D018749:RNA, Plant