RNA Injection Protocol

RNA Injection Protocol

This is an updated RNA injection protocol, since the one currently available is a little outdated and not detailed enough to help new people (or those less familiar and forced to do it during the COVID-19 pandemic). Tina and Dr. Norimatsu can always help, but Alec wrote this guide.

Video

Needed equipment:

1. The microinjector, glass micropipettes, binocular microscope, and light mineral oil (all of these should stay at the workstation).

2. Oocyte injection dish (typically at the workstation; the small plastic dish with hard plastic mesh for oocytes to stay in). Submerged in 70% Isopropyl alcohol (refill as needed)

3. MBSH and MBSH AB+AF, kept in the door of Fridge A.

4. Oocytes from incubator. Use the best quality oocytes you can find, typically in the most recent 12-well dish.

5. A 12-well dish for newly injected oocytes. You can use a partially filled one from the incubator or get a fresh one from the white box below the workstation. Only use the first and third columns (the second and fourth columns are used for sorting out the survivors after a few days).

6. Two micropipettors with tips: the 0.1-2.5 μL micropipettor with a box of red tips and a large micropipettor (can take up to 5 mL) with at least one large tip. The larger micropipettors and their tips can generally be found on the shelves between the two sides of the lab, behind the Frog Ringer and DI water jugs.

7. A small plastic transfer pipette. It's usually best to just get a fresh one instead of using one left there. Fresh pipettes can be found at the eye-level cabinet opposite the workstation.

8. A small beaker from the back corner of the workstation. This one's optional, but useful.

9. The yellow RNA cooler from Freezer A.

Procedure

1. Wear gloves. Scrub gloved hands with Eliminase until dry.

2. Take the green key and the yellow RNA cooler (in Freezer A) to the equipment room on the second floor. Get RNA out of the freezer for injection — the appropriate RNA should be in the CFTR 2020, TAAR1 2020, or Opioid Project boxes.

3. Rinse the oocyte injection dish with DI water. This will get out any oil residue, dust, or junk still present from the last injection.

4. Pour some normal MBSH from the large jug into the oocyte injection dish. If it's too full to pour, you can use the large micropipettor to incrementally fill the dish. Be sure there's enough to cover the whole dish and fully immerse the oocytes.

5. Use the transfer pipette to scrape the bottom of the injection dish. This will force MBSH into air pockets remaining under the mesh. Keep doing this until most or all of the air pockets are gone.

6. Select some good oocytes. You can try to do this under the microscope from the 12-well dish, or you can take a few out of each well and check them in the oocyte injection dish.

   1. Good oocytes are spherical, and have clear, high-contrast black and white poles. These should be injected.

   2. Bad oocytes can have flattened sides, darkened rings on the white pole, freckle-like dark spots on the black pole, regions of gray or green color, and/or less-than-expected contrast between the poles. These should be removed and flushed with water in the sink. If you got a small beaker, you can put the bad oocytes there temporarily so you don't have to get up as often.

7. This guide assumes the microinjector is already set up for injection, and that the dial is at 9.0 μL. Be sure the microinjector is set up for injection. If not, proceed to #18.

8. Make a fresh small mark with Sharpie on the plastic ruler at the back of the microscope. Use the small micro pipettor with a fresh tip to place 1 μL of RNA solution just below that mark. Put the ruler under the microscope and move the microinjector so that the tip of the glass micropipette is pointing toward the droplet.

9. Turn the silver dial below the microinjector clockwise (away from you) to move the microinjector toward the microscope stage. Reposition the ruler as necessary. Move the glass micropipette tip into the droplet, but not so far as to touch the ruler.

10. Fill the micropipette with the RNA solution by turning the orange microinjector dial counterclockwise (toward you). In theory, you should be able to fill with the full 1 μL, though this may prove difficult. Fill with at least 0.5 μL.

11. Turn the silver dial below the microinjector counterclockwise (toward you) to pull the microinjector back up to the starting position. Use a Kim-wipe to remove any remaining RNA solution from the ruler and set the ruler aside.

12. Move the injection dish onto the microscope stage. Use the silver dial to move the microinjector toward the oocytes. To impale an oocyte, slowly push the glass micropipette tip through the membrane at or near a perpendicular angle. This should form a dimple. Once you feel comfortably in, pull the microinjector slightly out, relieving the dimple slightly.

13. Turn the orange microinjector dial clockwise (away from you) to inject 0.05 μL of RNA solution. The final, small number of the injector readout should move from 0 to 5 (or 5 to 0).

14. Slowly turn the silver dial counterclockwise (toward you) to pull the micropipette out of the oocyte. Occasionally, a bit of cytosol and yolk will be in the tip of the micropipette. Eject it by turning the orange dial clockwise another 0.05 μL.

15. Repeat 12-14 for each oocyte. Once all oocytes are injected, use the large micro pipettor to put ~3 mL of MBSH AB+AF in a well in your injected oocyte dish. Then, use a transfer pipette to move the injected oocytes into the well. Label the well with the RNA injected, your initials, and the date.

16. Eject any remaining RNA solution from the glass micropipette into the oocyte injection dish. Continue ejecting (turning the orange dial clockwise) until an oil bubble forms at the tip, and then to a comfortable number for you on the dial. Use the silver dial to pull the microinjector back to the starting position.

17. Repeat steps 8 through 16 until finished. Clean up glassware, return oocytes to the incubator and RNA to the freezer, and dispose of bad oocytes in the drain.

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18. If the micro pipettor is not already set up, follow this protocol for it. 

19. Remove the micro pipette out of the stand, careful not to bend the metal rod at the end. Remove the tip and black rubber ring from the pipette and place them onto a Kim wipe, on the desk. Set the pipette at 9.0 μL.

20. Grab an injection pipette (preferably one that's been polished) and insert the unsharpened end into the black plastic tip, and then the rubber ring. The narrow end of the tip should point towards the sharp end of the injection pipette (also referred to as an injection pipette tip, glass pipette, or RNA pipette). 

21. Line up the sharp end of the micro pipette tip along with the end of the micro pipette's metal rod. Push the rubber ring down until it lines up with the notch on the side of the micro pipette. 

22. Holding the glass pipette in one hand, and the mineral oil syringe in the other, inject mineral oil into the blunt end of the glass pipette tip up until the level of the rubber ring. Be sure to continuously push down on the syringe while removing the needle from the glass pipette. If there are any air bubbles in the tip, you may be able to get them out by touching the blunt end of the tip to a Kim wipe repeatedly, or by inverting the glass pipette and flicking the blunt tip gently. If this doesn't work, the glass pipette tip is unusable and you will need to start again with a new one.

23. When you get a pipette tip that has no air bubbles, and the oil reaching the black rubber ring, you will gently insert the metal rod of the micro pipettor, being careful not to push any bubbles in. Allow the metal rod to fill the tip slowly by using gravity to your advantage. If you do this too fast, the pressure of the meta rod could blown out the glass tip, and you would need to start from scratch. 

24. When the rod is fully in, push the black plastic tip towards the handle of the micro pipettor, and screw it on. 

25. Turn the orange knob counter clockwise (towards the wall) to push the mineral out to the end of the tip. If the oil overflows down the side of the glass tip, you can gently touch the side of a Kim wipe to it. Do not touch the end of the tip with the Kim wipe. This will break your RNA injector tip, and you will need to start over. 

26. Prepare the microscope slide by wrapping it in a new piece of parafilm after disposing of the old piece. Do not touch the side of the parafilm that will make contact with the RNA. Mark around the area of the place you want to deposit the RNA (from where to draw it up from) with a fine tipped sharpie. 

27. Pipette around 1.5 μL of RNA (after thawing it) on to the slide, where you marked off for in step #26. This should form a small, clear droplet. 

28. Move the slide so that it's in the center of the field of vision of the microscope. Readjust the light if needed. 

29. Line up the micro pipette tip with the droplet, and while looking through the microscope, submerge the tip in the droplet. You will likely need to be at high magnification to see the tip actually penetrating the droplet's surface. 

30. One penetrated, look through the microscope as you turn the orange knob clockwise (towards yourself), making sure that the tip stays continuously submerged while turning the knob. If the tip comes out of the droplet while you are turning the knob, an air bubble will form. If an air bubble forms, turn the knob in the opposite direction to push it out, and start again

31. Get as much RNA as you can without touching the tip to the parafilm, and without pulling up air. You will not get all of the RNA and that's okay. Keeping from breaking the pipette tip is key here. 

32. When complete, withdraw the micro pipette and push the slide to the back of the microscope platform, and set up the mesh bottomed dish. 

33. Line up the oocytes in a convenient way that will make them easy to inject; in a horizontal line, or group them all on one side of the dish, and manually move them individually after injection, for instance. 

34. Impale the oocyte, watching for "the pop" of the tip breaking past the cellular membrane. 

35. Inject 0.05μL of the RNA. Remove the tip slowly, and move onto the next oocyte. If the oocyte does not come off, withdraw the glass tip from the MBSH solution. The surface tension of the fluid will pull the oocyte off of the injection tip.