This protocol uses Laser-Capture Microdissection coupled with RNA-Sequencing to obtain Spatial and Temporal transcriptomes from specific cells in plants of interests, using small quantities of biological materials. The main advantage of laser technique is that it facilitates the direct visualization of cells within normal tissue context, allowing the discrete cells to be precisely isolated in a contact free manner. Although this protocol is optimized for the isolation of plant cells, it can be applied to most cells that can be stologically identified.
Good sample preparation is critical. Therefore, one performing this technique for the first time, prop optimization of the tissue fixation and the embedding is important before Laser-Capture Microdissection. Before collecting the tissue sample, prepare a fixative appropriate to the species and tissue type to be harvested.
For barley seed, cut the seed samples in half before submerging the samples into at least a 10 X volume of ice cold fixative. Use vacuum infiltration to accelerate the penetration of the fixative. The tissue should sink by the end of the infiltration, then refresh the fixative for an overnight incubation and transfer the samples into cassettes for tissue processing.
Place the tissue loaded cassettes into the metal basket of an automatic processor and attach the metal basket to its holder above chamber one. Set the program on the control panels of the tissue processor and press start to begin the automatic processing program. The system will dehydrate the samples by dipping the cassettes into a gradient series of ethanol for 90 minutes per concentration as indicated.
After the last ethanol immersion, the system will submerge the samples in xylene gradients for 90 minutes per solution as indicated. Followed by 290 minute immersions in molten paraffin at 55 to 60 degrees Celsius. The next morning, remove the paraffin infiltrated cassettes from the tissue processor and proceed to the paraffin embedding.
The next morning, use fine forceps to transfer the process samples into suitably sized molds and add molten paraffin over each sample. For barley seeds, orient the samples in the paraffin longitudinally to the cutting direction to facilitate the acquisition of longitudinal sections. Place a clean cassette onto the mold and ensure that the entire cassette is fully covered with a sufficient volume of paraffin to secure the sample to the cassette.
Then place the mold onto a cold plate for 10 to 20 minutes until the paraffin is set before releasing the block from the mold. To prepare a PEN Membrane Slides, submerge the slides in RNS deactivating solution for three seconds followed by two brief washes and DEPC treated water. Then drive the slides in a 370 degrees Celsius incubator to remove any leftover solution and UV treat the slides in a laminar flow cabinet for 30 minutes to enhance their paraffin not hazelnuts.
For a sample tissue sectioning place the paraffin blocks on the cold plate and fill the water bath with the DEPC treated water, then warm to 42 degrees Celsius. Trim blocks to the depth of the region of interest and section the paraffin blocks to a six to 10 micron thickness. A well sectioned block will form a ribbon at the edge of the blade.
Use a fine tweezer to gently transfer ribbons from the microtome to the water bath, taking care that the ribbon lays flat on the surface of the water. To collect the sections, holding a slide at a 45 degree angle, use an upward motion to lift a ribbon out of the water onto the slide and use a lint free tissue to carefully remove the excess water. When all of the sections have been collected, wash the slides with 320 second washes in xylene followed by 230 seconds washes in 100%ethanol and 230 second washes in 70%ethanol.
To microdissect cells of interest from the deparraffinized and dry tissue sections, load slides on the three LCM microscope slots and load collection tube into the available slots. Move the stage to locate the region of the sample that needs to be cut and cut a blank segment free of tissue on the membrane slide. To optimize the cutting speed and the cutting in laser pressure catapulting energy and focus.
Use the drawing tools to outline the area of interest in the tissue and use the optimized parameters and the Robo LPC function to catapult cells into the adhesive caps. Select flag from the elements list to use the flag tool to mark the regions of interest. Check the cap check button to inspect the adhesive cap, to confirm that the samples have been captured.
Typically 10 to 15 sections per cap are required for RNA extraction. Immediately after all of the samples have been acquired, proceed immediately to RNA extraction to avoid RNA degradation. After RNA extraction and RNA amplification use an automated electrophoresis system, according to the manufacturer's instructions to quantify and qualify the antisense RNA.
In this study, LCM, RNA-sequencing was applied to a small number of cells from three embryo organs, every eight hours over a 48 hour time course during germination. It's important to adjust the cutting parameters correctly to allow the tissue of interest to be precisely cut and dislodged from the surrounding tissue without burning the edge of the selected area. Ramification of the total RNA before RNA amplification allows the detection of distinct electrophoretic bands and fluorescent peaks of 18 and 28S ribosomal subunits in good quality RNA samples.
Successfully synthesized antisense RNA will exhibit a unimodal symmetrical size distribution from 100 to 1000 nucleotides with a peak around 300 nucleotides after two rounds of amplification. Multi-dimensional scale plotting of genes expressed in the different tissues over 48 hours of germination, illustrates a greater similarity between samples of a single tissue than between samples from the same time point, but different tissues. The number of differentially expressed genes increases progressively over the course of germination in each tissue relative to the zero hour time point of the tissue.
In this representative analysis, 25%of plumule, 34%of radicle and 41%of scutellum differentially expressed genes were found to be exclusively expressed within each tissue. It is important to correctly adjust the cutting parameters for a precise excision and dislodging of the selector area from the surrounding tissue without burning the edge of the selected region. With improved chromatin and protein purification methods, and enhance mass spectrometry instrument, LCM can be used for cell type specific epigenomic and proteomics studies implants.
