Laser microdissection of tomato fruit cell and tissue types for transcriptome profiling

Abstract

This protocol enables transcriptome profiling of specific cell or tissue types that are isolated from tomato using laser microdissection (LM). To prepare tissue for LM, fruit samples are first fixed in optimal cutting temperature (OCT) medium and frozen in molds. The tissue is then sectioned using a cryostat before being dissected using an LM instrument. The RNAs contained in the harvested cells are purified and subjected to two rounds of amplification to yield sufficient quantities of RNA to generate cDNA libraries. Unlike several other techniques that are used to isolate specific cell types, LM has the advantage of being readily applied to any plant species without having to generate transgenic plants. Using the protocols described here, LM-mediated cell-type transcriptomic analysis of two samples requires ∼8 d from tissue harvest to RNA sequencing (RNA-seq), whereas each additional sample, up to a total of 12 samples, requires ∼1 additional day for the LM step. RNA obtained using this method has been successfully used for deep-coverage transcriptome profiling, which is a particularly effective strategy for identifying genes that are differentially expressed between cell or tissue types.

High-resolution spatiotemporal transcriptome mapping of tomato fruit development and ripening

“…The five pericarp cell/tissue types (outer epidermis, collenchyma, parenchyma, vascular tissue, and inner epidermis) were harvested by LM from equatorial sections of fruit at 5 DPA, 10 DPA, 20 DPA, MG, Br, Pk, LR, and RR stages. Samples for LM were fixed, cryoprotected, embedded in optimal cutting temperature (OCT) medium, and frozen in molds using liquid nitrogen, whereas those collected at Pk, LR, and RR were directly embedded in OCT without fixation/cryoprotection, to preserve their integrity 70 .…”

“…Sections of embedded tissues for the LM samples were mounted on adhesive-coated glass slides using a CryoJane transfer system (Instrumedics) 70 , except the anthesis and 5 DPA samples, which were mounted on PET-membrane 1.4-μm slides (Leica). Section thickness was set at 12 μm for membrane slides and 16 μm for glass slides.…”

The Tomato Expression Atlas

“…Large-scale transcriptome profiling has become widely adopted as a means to characterize the status and diversity of biological samples, and as a platform for functional genomic studies. Such analyses typically target whole organisms or specific organs; however, there is an increasing interest in using cell- or tissue-related transcriptome profiling to provide enhanced spatiotemporal understanding of gene function ( Martin et al , 2016 ). The quantity and resolution of such gene expression information necessitates the development of new data visualization tools that are more interactive, intuitive and accessible.…”

Long-Term Impact of Chemical and Alternative Fungicides Applied to Grapevine cv Nebbiolo on Berry Transcriptome

“…As a first result, a protocol to apply this technology to collect separately two different cell-type populations grape berry skin was obtained. LMD technology was already used to dissect the complexity in citrus and tomato fruits [ 51 , 52 ]. In grapevine, LMD was applied to analyze the transcriptional changes in stomata cells and surrounding areas of grapevine leaves at early stages of Plasmopora viticola infection [ 53 ], to identify the site-specific gene responses in grapevine leaf phloem infected by stolbur [ 54 ] as well as to verify the involvement of vessel-associated cells in embolism recovery in leaf petiole [ 55 ].…”