Isolation and Quantification of Axonal mRNAs Using Porous Membrane Inserts and RTddPCR

Our research studies how local protein synthesis is regulated and their roles in neural repair, development, and degeneration. Recent advances include high-sensitivity mRNA detection techniques, like gdPCR, for identifying low-abundance axonal MRNAs, and compartmentalize neuronal cultures. To begin aliquot 250 microliters of triazole into a 1.5 milliliter micro centrifuge tube corresponding to each insert into one well or insert of a six-well plate.

Keep tubes aside until needed. Add two milliliters of sterile PBS into each well of a second six-well plate, ensuring the number of wells or plates matches the number of inserts. Pipette out the culture media from both the top end bottom of the insert and transfer the insert into the six-well plate containing PBS.

Now, using forceps, gently place the insert, then add two milliliters of PBS on top of it. Aspirate the PBS from both sides and repeat to wash twice. Then leave the insert in fresh PBS.

Next, use a sterile cell scraper to scrape the whole neuron fraction from the top of the insert. Collect the soma lysate from the insert. Transfer it into a 1.5 milliliter micro centrifuge tube.

Centrifuge the tube at 10, 000 to 15, 000 G for two minutes. Discard the supernatant and resuspend the pellet in 250 microliters of trizol. Label this tube as the whole neuron fraction.

To collect the neurite fraction, move one end of a sterile cotton swab slowly in a zigzag pattern from top to bottom on the whole neuron side of the insert. Rotate the insert 90 degrees and repeat using the other end of the swab. Then discard the swab.

Use a new swab and move in concentric circles, starting from the center of the insert outward, making sure to clean the circumference as well. Invert the insert so the neurite side faces up. Cut the membrane using a new sterile scalpel blade.

Place the cut membrane into the six-well plate containing trizol with the neurite side facing down. Ensure that the membrane is submerged. Now collect the trizol containing the neurite lysate from the well.

Transfer it into a 1.5 milliliter micro centrifuge tube. Proceed with RNA isolation or store the soma and neurite lysates at minus 80 degrees Celsius for later processing. Prepare reverse transcription droplet digital PCR reactions using Droplet Digital PCR Ready-To-Use Universal Mix and target specific primers with appropriate complimentary DNA.

Pipette the reaction mixture into the droplet generation cartridge. Then add the generation oil into the designated wells of the cartridge. Now, seal the cartridge with the gasket.

Generate the droplets using the droplet generator. Once the droplets have been generated, transfer them into a 96-well PCR plate and seal with foil before placing the plate in the thermocycler. Open the QX Manager Software to begin the analysis.

Click on the Browse option and open the file to be analyzed. When the file has loaded the dashboard view appears a on the left panel. Select the wells of interest by holding the Control key while clicking.

Click on 1D Amplitude to visualize a dot plot. Select Threshold Multiple Wells to apply the same threshold to more than one well. Enter the threshold value based on where a clear separation is seen between positive and negative droplets.

Now view the data section well, showing the sample description, concentration values, number of accepted droplets, as well as positive and negative droplet counts. Click Save to record the thresholding results. RNA quantification using the ribo green assay revealed that whole neuron fractions yielded 212.85 nanograms of RNA per insert while neurite fractions yielded 42.75 nanograms.

PCR validation identified primer set one as the most specific for the Gap43 transcript, showing a distinct single band. Ambiguous PCR results for Gamma-actin prompted a temperature gradient test using primer set two, which showed strongest amplification at 55 degrees Celsius. RT-ddPCR amplitude plots showed clear droplet separation for Gamma-actin in both whole neuron and neurite samples, confirming reliable transcript detection.

For Gap43, nearly 23%of the mRNA pool is present in the neurites, as compared to Gamma-actin, where only 5%of the mRNA pool is present in the neurites compared to the whole neuron fraction. We have shown the presence of stress granule structures in the axons under physiological conditions, which inhibit local protein synthesis. Our protocol offers a reliable and consistent method for isolating neuronal compartments and detecting low-environment mRNAs.

Future analysis will focus on isolating and characterizing distinct axonal sub-domains, such as growth cones and axonal shaft beyond bulk analysis.