Single-Animal, Single-Tube RNA Extraction for Comparison of Relative Transcript Levels via qRT-PCR in the Tardigrade Hypsibius exemplaris

We are focused on uncovering the molecular mechanisms of neuroresilience in the extremotolerant tardigrade Hypsibius exemplaris. We study nervous system changes after exposure to extreme environments. We have discovered large-scale changes to neuroanatomical structures and motor function in tardigrades after they have experienced extreme environments.

The use of tardigrades as a model organism for molecular biological research is relatively new. As such, there are many techniques that are not yet fully optimized for their application in tardigrades. Though, we have made remarkable strides in transcriptomics, proteomics, and transgenesis, there are still methods and tools required for the verification and functional assessment of target proteins that remain underdeveloped.

Current protocols for RNA extractions from single tardigrades have not yet been applied to qPCR or require multiple animals for extractions. To continue our work on the molecular neurobiology of the tardigrade, we needed a way to extract and quantify RNA derived from individual animals to first, confirm RNA sequencing findings, second, assess RNAi efficiency in individually injected animals, and three, explore individual variances in transcriptional changes across populations. Our method improves upon previous techniques in three ways.

It decreases the time of extraction by approximately 30%as compared to previous single tardigrade extraction methods. Our method also decreases the cost of extractions by over 100 fold as it bypasses the need for linear PCR amplification step. Finally, our method is 200 fold more efficient than previous methods as it bypasses the need for a purification, which often results in significant loss of RNA.

Our findings will allow tardigrade researchers to quantify relative transcript levels via qRT-PCR from individual tardigrades in a low cost and efficient manner. We see this being used to quantify the efficiency of RNAi knockdown in a highly quantitative manner, confirm sequencing hits, and explore individual differences in transcriptional response across various animals. To begin, light a Bunsen burner or another controlled flame source at a low setting.

Hold a glass micropipette with one end in each hand and position its center over the flame until the glass begins to melt. Once softened, rapidly pull the two ends apart to create two sharp tips. Then, using a pair of sterile fine forceps lightly break the tip of the micropipette to adjust its sharpness.

Secure a glass micropipette on a micropipette puller, avoiding contact with the heating filament. As a starting point for optimization, set the polar to 78 degrees Celsius with a single pull step using a pull weight of 182.2 grams. Let the filament heat, causing the glass micropipette to separate into two micropipettes with sharp points due to gravity.

Store the pulled glass micropipettes in a closed 100-millimeter Petri dish and secure them with wax or clay to prevent the sharp tips from breaking. To begin, obtain the pulled glass micropipettes of desired dimensions. Then prepare the complementary DNA synthesis Master Mix and tardigrade lysis buffer.

Aliquot sufficient lysis buffer for extractions using two microliters per tardigrade. Add ribonuclease inhibitor to the lysis buffer to achieve a final concentration of four units per microliter. Vortex the solution briefly, then spin it down in a tabletop centrifuge at 2000g for five seconds at room temperature.

Store the solution on ice. Using a sterile filter tipped P1000 pipette remove the required number of tardigrades from the culture and place them in a sterile 35-millimeter Petri dish. Wash the tardigrades three times with one milliliter of autoclaved sterile spring water to remove algal contaminants.

Place the dish under a dissecting microscope with 25X to 50X magnification and use a filter tipped P10 pipette to transfer a single tardigrade to a new sterile 35-millimeter Petri dish. Now, with a sterile filter tipped P200 pipette wash the single tardigrade in 100 microliters of sterile nuclease-free water. Transfer the washed tardigrade to a clean sterile PCR tube in one to two microliters of sterile nuclease-free water.

Visualize the tardigrade under a dissecting microscope at 25X magnification. To facilitate water removal, break the tip of the pulled glass micropipette lightly outside of the tube. Using the glass micropipette's capillary action remove water until the tardigrade is surrounded by a small bubble of water.

Add two microliters of tardigrade lysis buffer to the tube. After vortexing briefly centrifuge the tube at room temperature for five seconds at 2000g and store the sample on ice. Then place the sample containing tardigrades into a PCR tube rack and secure it tightly.

Gripping the rack with long coarse forceps gently immerse it into liquid nitrogen until the sample is fully frozen. Next, remove the rack from the liquid nitrogen and place it on ice. Thaw the sample, monitoring it every 15 seconds until it turns visibly transparent.

After repeating the freeze-thaw process five times, place the sample on ice. Add two microliters of complementary DNA synthesis Master Mix to the PCR tube containing tardigrade lysate. Briefly flick the tube and spin it at 2000g for five seconds at room temperature and place the sample on ice.

Now, load the sample into a thermocycler and start the cDNA synthesis program. After the synthesis is completed immediately place the tube on ice. Dilute the sample to a total volume of 25 microliters with 21 microliters of sterile nuclease-free water.

The optimized protocol achieved a threefold increase in complementary DNA yield compared to protocols without freeze-thaw cycles, with six cycles being optimal. RNA extraction was unsuccessful when residual water was present, demonstrating the critical need to remove excess water for consistent lysis. The RNA yield was around 14.24 nanograms per tardigrade.

This protocol was over 200 fold more efficient than RNA extraction kits in actin transcript yield, as demonstrated by significantly lower Ct values and robust gel band amplification.