Laser Microdissection for Species-Agnostic Single-Tissue Applications

We combine laser capture microdissection with a single cell RNA seek protocol called CEL-Seq2 to generate transcriptomic data sets for small individual tissue samples. This technique lets us study gene expression in tissues or species that cannot be studied using traditional cell sorting methods. Additionally, it provides more specificity than a bulk RNA-seq approach.

Here we demonstrated this technique for the four celled tail tips of C.elegans fourth larval stage males and hermaphrodites. But the beauty of this approach is that it can even be applied to non-model species. Demonstrating the procedure will be Ms.Raya Jallad, PhD student from my laboratory and Dr.Antonio Herrera, currently lead biomedical scientist at the Baylor school in Chattanooga, Tennessee.

Begin by gently pipetting one to two milliliters of M9 buffer against the wall of the plate without squirting. Swirl the plate to dislodge the worms. Remove and discard all the liquid and the worms by placing the pipette tip against the wall of the plate at the edge of the agar to avoid poking holes.

This video shows the plate before mothers and larva are removed. Only embryos should be left after the mothers and larva are removed. L1 larva will start appearing after incubation for an hour or so.

Then place the plate at 25 degrees Celsius. After one hour, remove the plate from the incubator. Carefully drop one milliliter of M9 buffer onto the agar and swirl the plate to dislodge the L1s but not the embryos.

Centrifuge the tube and pipette the L1s directly onto the bacterial lawn of a seeded plate. Keep the worms at 25 degrees Celsius. Under a dissection microscope at 30 to 50 X magnification, begin picking the males and the hermaphrodites from the synchronization plates onto separate unseated plates.

Males are distinguished from hermaphrodites by the bulged shape and lighter color of the male tails compared to the pointed shape and darker color of the hermaphrodite tails. Wash the worms off the plate with one to two milliliters of M9 buffer using a pipette tip pre-washed with M9 buffer containing 0.01%detergent. Transfer the worms to a one milliliter centrifuge tube, spin for one minute and add one milliliter of M9 buffer.

Again, spin for one minute. Add one milliliter of ice cold 70%methanol and mix well. Repeat the wash with one milliliter methanol, mix well.

Spin it for one minute and add 500 microliters of 70%methanol. Mix it and store it at four degrees Celsius for one hour to overnight. Pipette 20 microliters of the fixed worms onto the coated side of a polyethylene naphthalate or PEN-membrane glass slide.

Wait for the methanol to evaporate. Remove the plastic shield over the stage and click the unload button with the upward arrow for loading the membrane slides. Make sure the slide is completely dry, flip so that the membrane is facing down and insert the slide.

Click continue in the change specimen window. The slide holder will move. Then replace the plastic shield, on the bottom of the screen, choose which slide holder contains the slide and click the unload button with the downward arrow to load the tubes.

Pull the tray out and remove the tube block. Insert the tube caps of four 500 microliter PCR tubes into the holder and fold the tube under. Return the block to the tray and slide the tray back into the microscope stage.

In the change collector device popup window, select PCR tubes, and click okay. Click on the empty tube location on the bottom left of the screen under collector device tube caps and in the microscope control panel select TLBF for transmitted light bright field illumination. Using the 2.5X lens, adjust the focus until the worms and the surface structure of the PEN-membrane are visible.

Switch to the 20X lens and move the stage to a region without worms. Adjust the focus such that the bubble like structures in the membrane have a yellowish color to focus the laser on the correct focal plane then set the laser parameters. For the tail tips, start with power 45 aperture 30 in speed 20.

In the laser control panel, select calibrate and follow the instructions. Next, on the bottom of the screen at collector device tube caps click on position A, on the right side of the screen select single shape, followed by draw and cut. Then select point to point and draw a line.

Click start cut so that the laser cuts through the membrane. Find a worm and switch to move and cut. Use the mouse to cut through the tail.

To collect the sample, switch to the draw and cut setting with the point to point function and draw shape to complete the cut of a membrane section. Select the next tube at collector device tube cap on the bottom of the screen and cut the next tail tip. Once four tails are cut, unload the tube rack by clicking unload with the downward arrow.

Find the membrane sections under a dissecting microscope and continue with the downstream sample processing. Here RNA sequencing with CEL-Seq2. Pipette 1.2 microliters of a CEL-Seq2 primer master mix directly on top of the sample and close the tube.

Label with the primer number and immediately place the tube cap directly on a piece of dry ice to flash freeze the sample thus preventing RNA degradation. Repeat until all samples have been collected. Finally store the tubes at minus 70 degrees Celsius.

C.elegans L3 hermaphrodites and males at 21 to 23 hours after hatch can be distinguished under a dissection microscope by the morphology of their tails. The tale of hermaphrodite is narrow while that of males is swollen and appears clear. The appearance of the PEN-membrane slide structure and worm tail is shown in these images.

Here the focus is correct for the 20X and 40X lenses at the microscope. The dissected tail impartially cut out PEN-membrane are visible here. After closing the gap in the cut, the membrane piece will drop into the tube cap below the slide.

A tube cap with a PEN-membrane section containing a dissected tail tip is shown here. The graphical image represents the natural log transformed unique molecular identifier or UMI counts per individual tail tip for different time points and sexes. The powsimR software is used to determine how many independent samples are required to detect differential expressed or DE genes at various expression levels.

The graphical image here represents the true positive rate to detect the DE genes between two conditions for four different simulations incorporating different sample sizes per condition. The dashed line indicates an 80%true positive rate. The false discovery rate and the same four simulations is shown here.

The dashed line indicates a 10%false discovery rate. The graphs showed that a sample size of 70 tail tips per condition is sufficient for detecting the DE genes except for the genes with very low expression levels. For proper synchronization, ensure that only embryos are present on the plate.

To ensure against sample loss, pipette the primer mix directly onto the PEN-membrane section in the cap and be extremely gentle in closing the cap. Because it's species agnostic, we can use this protocol to explore how gene regulatory networks have evolved for homologous structures in different species.