Microinjection of dsRNA in Tardigrades

1. Amplify the region of interest from genomic DNA or cDNA in a PCR that attaches T7 polymerase binding sites to the 5′ end of each strand.

• We have had success with PCR products in the range of 275–1000 bp.

2. Use 1 µg of the final DNA product from Step 1 as the template to synthesize dsRNA via an in vitro transcription kit, following the manufacturer's directions.

3. To purify the dsRNA, add 0.1 volume of 3 m sodium acetate and 2.5 volumes of 95% ethanol to the sample from Step 2. Incubate on ice for 5 min and then centrifuge at maximum speed in a microcentrifuge for 10 min.

4. Remove the supernatant and then wash the pellet with 0.5 mL of ice-cold 70% ethanol. Centrifuge at maximum speed in a microcentrifuge for 5 min. Remove the supernatant.

5. Air-dry the pellet at room temperature, and then resuspend the dsRNA in nuclease-free H₂O.

6. Determine the concentration of dsRNA via spectrometry.

• The extinction coefficient for dsRNA is 45.

7. Store the dsRNA solution at −70°C.

8. Use a micropipette puller to pull needles to a fine taper from borosilicate glass tubing (1.0-mm OD, 0.58-mm OD). Follow the manufacturer's instructions to obtain needles that taper quickly into a sharp point, with a bore of about 1 µm.

• To achieve a good needle, it may be necessary to adjust the pulling force and/or the temperature of the heating filament. If the temperature and/or the pulling force are too low, then the needle may have a long taper that is too flaccid to penetrate the tardigrade's cuticle. Conversely, if either parameter is too high, then the needles could result in a bore size that is too large for injections.

9. Store the needles on a long piece of clay molded into the bottom of a 100-mm Petri dish and keep covered until needed.

10. For microinjection experiments, dilute the dsRNA to a final concentration of 1 µg/µL in nuclease-free H₂O. Centrifuge briefly to clear the supernatant before filling the injection needle.

11. Back-fill the injection needle with dsRNA, and allow the dsRNA to flow to the tip of the needle by gravity.

12. Prepare the injection chamber as follows.

• i. Use a glass cutter to remove the corner of a microscope cover glass (22 mm × 22 mm, No. 1½).

• ii. Place the triangular corner piece in the middle of a standard glass microscope slide, such that one straight edge is parallel to the short edge of the slide (Fig. 1).

• iii. Secure the cover glass to the microscope slide with clear nail polish.

  • The purpose of the cover glass is to create a solid barrier against which to brace the animals. Imperfections in the cover glass can sometimes create a gap between the cover glass and the microscope slide large enough for the animals to slip under. Mounting corners of cover glass, rather than a whole piece, appears to result in fewer gaps.

  • Chambers can be prepared in advance and stored in a microscope slide box.

13. Using a stereomicroscope, transfer adult tardigrades into one well of a deep two-well depression slide. Allow animals to excrete any material from their intestines, changing the spring water several times.

• This step can take up to 30 min.

14. (Optional) Anesthetize the animals in levamisole immediately before performing injections by replacing the spring water with 10 mm levamisole (prepared in spring water).

• This step helps to minimize animal movement during microinjections (Tenlen et al. 2013) but is not required (Boothby et al. 2017).

15. Apply a drop of Halocarbon 700 oil to the straight edge of the cover glass on the injection chamber.

16. Using a stereomicroscope, transfer several animals to the oil drop on the prepared injection chamber. Position each animal lengthwise along the straight edge of the cover glass. Remove any residual water.

• We typically inject 5–10 animals at a time; animals appear to be unaffected by exposure to halocarbon oil.

17. Mount the microinjection needle in the needle holder of the micromanipulator.

18. Position the injection chamber on the inverted microscope and orient the micromanipulator so that the needle is perpendicular to the tardigrade (Fig. 1). Under the lowest-power objective, bring both the animal and needle in focus.

19. Switch to the 20× objective and lower the needle into position. To break the needle, use the micromanipulator controls to bring the needle into the same focal plane as the cover glass edge, then gently brush (stroke) the needle against the coverslip edge.

20. Using the micromanipulator fine controls, adjust the needle so that it is in the same focal plane as the animal. Bring the needle in contact with midpoint of the body (so that it is targeting the gonad/intestine) and then gently tap on the left side of the microscope stage to force the needle into the body.

• Depending on the bore size, one or two taps of the foot pedal should be sufficient. Delivery of dsRNA can be confirmed by slight swelling at the site of injection.

21. Use the micromanipulator controls to withdraw the needle, and move to the next animal.

22. After all animals on a slide have been injected, move the slide back to the stereomicroscope. Add a drop of spring water over each animal, and then transfer the animals to the second well of the depression slide.

23. To monitor individual animals and their progeny, prepare a 96-well tissue culture plate (untreated, sterile) by adding 100 µL of spring water and 5 µL of green algae to each well. Transfer one animal to each well.

• Animals may instead be transferred in groups to 35-mm Petri dishes, with a layer of spring water and one to two drops of green algae.

24. Maintain all injected animals at room temperature, and monitor water and food levels regularly.