The overall goal of this protocol for LAM is to isolate specific plant cells for the generation of high-integrity RNA for transcriptional analysis. The focus is on inaccessible cells, like egg cells, in sexual and apomictic boechera species. This method can help answer key questions in the field of plant developmental genetics, such as the transcriptional basis underlying sexual and asexual plant reproduction through seeds.
The main advantage of this technique is that certain types specific styles can be perform. Overcoming the effect of masking transcriptions of rare cell types in the analysis of atherogenesis tissues. To begin this procedure, using the blocking station fill a pre-warmed, fresh plastic balancing tray with melted paraffin wax at 56 degrees Celsius.
Lift the lid of the paraffin bath and pick up one cassette at a time. Transfer a sample to the 56 degree Celsius liquid paraffin wax in a balancing tray. Proceed quickly to avoid any wax hardening around the sample while handling the cassette.
Then, position the sample according to the experimental design using a preparation needle. Repeat the procedure until all the samples are transferred. To prepare thin sections for LAM, on a lighted table, remove the paraffin wax with a sample from the plastic tray.
Dissect out a wax block containing the sample, such as a bud or flower, using an RNase-free razor blade. Next, melt a small droplet of parffin wax on the tip of a spatula. And use the hot wax to fix the block with embedded sample on the support.
Remove the extra wax surrounding the sample with a razor blade, and make sure the block has a square surface with parallel edges. Then, set the heating plate to 42 degrees Celsius. Safely mount the sample to the microtome.
Prepare seven micrometer thick sections for the cells of boechera mature gametophyte. Afterward, transfer the 10 to 15 centimeter long paraffin ribbons to a plastic box with black cardboard at the bottom before positioning them on the LAM slides. To prepare the slides for LAM, place five to ten metal-framed slides on the heating plate with the flat surfaces facing up.
Pipette one to two milliliters of RNase-free water on the plastic part of the slides. Using a razor blade, cut the paraffin ribbons in short pieces of about four to five centimeters. Long enough to span the plastic windows of the slides.
Then, use a pair of tweezers and a preparation needle to places the paraffin strips parallel to each other on the plastic window of the slides. Carefully lift the slides at one end to remove the water and place them back. Cover the heating plate with a plastic lid without touching the slides, and ensure that the plastic lid allows air exchange.
Dry the slides at 42 degrees Celsius overnight. Under a chemical hood, de-wax the slides in xylol two times for 10 minutes each time. Make sure to submerge only the plastic part of the slides and keep the broader end of the metal frame of each slide dry for removal.
Allow the slides to dry for 10 minutes in the chemical hood prior to LAM. In this procedure, turn on the computer and the microscope for LAM. Then, start the program steering the microscope and laser.
Turn on the laser control box and switch on the laser by pressing the corresponding button on the control box. Next, place a cap into the cap holder. Place the LAM slide on a glass slide from microscopy and ensure that the flat surface of the slide with the tissues attached faces the glass slide.
Then, place the slide in the instrument with the glass slide underneath. At last, position the cap holder with the cap in the instrument. Select the 4x objective.
In the program move the cap in the up position. Afterward, make a scan of the slide. Search through the slide and identify the structures of interest.
Pinpoint and save their positions on the slide to allow returning to the exact position for the cutting step. Then, select an appropriate objective. Verify the laser position by pressing the laser shot button once.
Ideally, in a part of the membrane without tissue. With the cap in the down position, use the hand pen tool to mark the borders of the cell type or tissue of interest. For example, the egg cell.
Dissect it with the laser by pressing Cut. In order to isolate individual cells, it is critical to clearly identify them in the right sections. Also, the cells should have a diameter of at least eight micrometers to make a cell type specific transcription analysis feasible.
Please note that sectioning needs to be repeated if the border around the cell has not been entirely dissected, which can typically be seen on the screen. In this case, it is recommended to set the software to automatically run two repeats of the section as a standard. After that, lift the cap and move to the next save position with a structure of interest and dissect the next cell section until all the cells of interest from one slide are collected.
Make sure the cap is positioned in a way that the new section sticks to an empty position on the cap. After the cell types of interest from all the slides have been isolated, remove the slide from the instrument. Place the cap holder in the down position and visually inspect the cap surface.
And use the 4x objective to exclude unspecific contaminations of the sample. Then close the cap. And freeze the dry sections at minus 80 degrees Celsius until further use.
Here is the schematic drawing of the mature female gametophyte. These are the thin sections through ovules harboring female gametophytes in boechera divaricarpa in which egg cells are clearly identifiable due to the characteristic morphology of the female gametophyte. Similar RNA quality was obtained as analyzed from the egg cell sections compared to the larger tissue areas harvested from the same slides as controls.
The RNA quality achieved was suitable for cell type specific RNA sequencing, leading to the identification of 236 genes expressed in the apomictic egg cell but not in the apomictic central cell, synergent cell, or apomictic initial cell. Nor in the cells of the mature sexual gametophyte of arabidopsis thaliana. Other cell and tissue types from different species can be profiled in order to answer additional questions of developmental genetics.
Such as the molecular basis of imprinted in the endosperm and embryos of plant seeds. After its development, this technique paves the way for researchers in the field of developmental genetics and plant reproduction to explore the transcriptional basis of apomictic reproduction in the genus Boechera. After watching this video you should have a good understanding of how to obtain good quality RNA for cell type specific transcription analyses using laser-assisted micro-dissection.
