Laser capture microdissection of plant cells from tape-transferred paraffin sections promotes recovery of structurally intact RNA for global gene profiling.
Laser capture microdissection and related technologies permit the harvest of individual cells and cell types. Isolation of either nucleic acids or proteins from laser-captured cells supports such downstream applications as the construction of cell-specific cDNA libraries and the profiling of expressed genes and proteins. The success of these endeavors is dependent upon the yield, purity and structural integrity of the macromolecules derived from harvested cells. Here, we report protocols that promote the isolation of structurally intact RNA from laser-captured cells of paraffin-embedded tissues. The use of a tape transfer system that obviates the need to wet paraffin sections prior to slide mounting significantly increases RNA structural quality. Integrity is assessed directly via electrophoretic separation of picogram-nanogram levels of total RNA isolated from multiple cell types, including those comprising Arabidopsis ovules, replums and stamen abscission zones. RNA prepared from specialized cells within siliques provided targets for profiling the Arabidopsis genome during replum cell development. Digital northern analysis of transcripts expressed near the threshold of the system's ability to score signal presence suggests that low-abundance transcripts representing as little as approximately 0.002% of total mRNA can be reliably detected. Microarray data reveal a significant shift from primary cell-wall metabolism to lignin biosynthesis in replum tissues during fruit maturation.
Cai
Suqin
S
Department of Horticulture, Iowa State University, Ames, IA 50011, USA.
Lashbrook
Coralie C
CC
D017360:Arabidopsis / Q000166:cytology* / Q000235:genetics*; D029681:Arabidopsis Proteins / Q000235:genetics; D020869:Gene Expression Profiling; D007834:Lasers; D042282:Microdissection / Q000379:methods*; D016612:Paraffin Embedding / Q000379:methods*; D018749:RNA, Plant / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*
D029681:Arabidopsis Proteins; D018749:RNA, Plant