Laser capture microdissection of plant cells from tape‐transferred paraffin sections promotes recovery of structurally intact RNA for global gene profiling

Abstract

SummaryLaser 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 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.

MicroRNA156: A Potential Graft-Transmissible MicroRNA That Modulates Plant Architecture and Tuberization in Solanum tuberosum ssp. andigena

“…For histology, a modified protocol of Cai and Lashbrook (2006) was followed. Briefly, leaves and stems of 8-week-old plants (the wild type and miR156 OE lines 5.1 and 6.2) grown under LD conditions were fixed in chilled ethanol:acetic acid (3:1; Merck).…”

Application of Laser Microdissection to plant pathogenic and symbiotic interactions

“…nucleic acids, proteins and/or metabolites) from the harvested cells. The preparation of plant samples has been clearly illustrated in several original papers (Asano et al 2002;Kerk et al 2003;Klink et al 2005;Inada and Wildermurth 2005;Tang et al 2006;Cai and Lashbrook 2006) and in several reviews (Day et al 2005(Day et al , 2006Nelson et al 2006;Balestrini and Bonfante 2008;). Two methods have been utilized to prepare sample sections for LM: cryosectioning and paraffin sectioning.…”

AGAMOUSTerminates Floral Stem Cell Maintenance inArabidopsisby Directly RepressingWUSCHELthrough Recruitment of Polycomb Group Proteins

“…Laser capture microdissection was performed as described (http:// seedgenenetwork.net/arabidopsis#procedure; Cai and Lashbrook, 2006;Hsieh et al, 2011) using the Arcturus laser capture microdissection instrument (Applied Biosystems). In brief, inflorescences from Ler and ag-1 plants were fixed with ethanol/acetic acid, dehydrated, and embedded in paraffin blocks.…”