Tribolium pRNAI: Pupal Injections (based on Posnien et al 2009 – CSH Protocols)
Updated 7/25/22 by Patricia Graham
1) Prepare large petri dishes, one for each dsRNA you plan to inject.
a. Add enough PRE-SIFTED whole wheat flour to cover the bottom of the dish.
b. Add 5% w/w yeast and stir.
2) Prepare slides with toupee tape
a. Line the tape up with one edge. Do not go to the very ends of the slide with the tape or you will find your fingers stick to it and jerk the slide around when you are working.
b. You can usually fit about 15 pupae on one slide, so make two slides for each dsRNA you want to inject.
3) Collect pupae from jars about 5 weeks old at 30oC.
a. Note that the pupae will wiggle when you pick them up.
b. Try for pupae that are older – darker wings and eyes. They survive better.
c. Collect about 50-60 pupae for each dsRNA you plan to inject. That will give you 20-30 females.
d. Sort females from males (see http://www.ars.usda.gov/Research/docs.htm?docid=12892). Put 6-8 male pupae in the middle of the flour in each petri dish to mate with your injected females.
4) Collect your dsRNA at an appropriate concentration (1-4 ug/ul) in injection buffer plus dye, and keep it on ice. 5ul of RNA is enough to inject 15-20 pupae.
5) Place the female pupae on the tape – ventral side up. Stick the very posterior of the pupae to the tape. Attach as little of the posterior end as is feasible (usually only one segment), or they will not eclose properly. I find using forceps to position them on the bit of slide that isn’t covered with tape and using a blunted needle to push on the abdomen works well. They can be stationed fairly close together.
6) Take the following with you to the injection room.
i. a 1-10 ul pipet,
ii. a needle (or 2) for each dsRNA you want to inject
iii. your plates with flour
iv. the slides with pupae
v. RNA on ice
7) In the injection room
a. Turn on the nitrogen
b. Turn on the microscope light
c. Turn on the pump
d. Using the 1-10 pipet and a loading tip (extra long and slim) load a needle
i. Push the tip FIRMLY onto the pipet
ii. Suck up RNA (Be patient. It’s a little viscous.)
iii. Push the end of the pipet tip to the bottom of the needle
iv. Push slowly to eject the RNA. Move the end of the pipet tip out of the solution carefully so as not to introduce bubbles.
e. Place the needle in the injection apparatus
i. Raise the apparatus before inserting the needle
ii. Insert until you can see the back end of the needle, then tighten.
f. Open the needle (Note - we now have a beveller, so you can open the needles using the beveller before injecting.)
i. Put one of the slides on the stage near the needle.
ii. Lower the needle down until it is below the top edge of the slide.
iii. Push on the foot pedal until the RNA is near the tip.
iv. Move the needle carefully in and rub the tip gently along the edge of the slide.
v. Push on the foot pedal. If nothing comes out, rub the tip again. Repeat until a drop comes out when you press the foot pedal (Note – a small drop is nice because you do less damage to the pupa when injecting, but the needle will probably clog during the course of the injections. Just repeat the steps here when it does, then go back to injecting.)
8) To inject
a. Raise the needle up above the slide.
b. Move the slide in until the needle is above the first pupa
c. Lower the needle until it touches the pupa
i. Be ready for the pupa to move.
ii. Aim just to the right or left of the midline at the join between the 3rd and 4th abdominal segments (about where the wing tips point).
iii. Slide the needle quickly in, then pull back just a little before pushing the foot pedal. If nothing comes out, slide the needle back and forth a couple of times and try again. If still nothing comes out, re-open the needle.
iv. Slide the needle out and raise it a bit before moving to the next pupa
9) When all pupa that can be are injected, remove the ones that didn’t get injected. Place the slide with the injected pupa face down into the flour. Keep in the 30oC incubator (be sure there is a beaker of water in the incubator too.).
Updated 7/25/22 by Patricia Graham
1) Prepare large petri dishes, one for each dsRNA you plan to inject.
a. Add enough PRE-SIFTED whole wheat flour to cover the bottom of the dish.
b. Add 5% w/w yeast and stir.
2) Prepare slides with toupee tape
a. Line the tape up with one edge. Do not go to the very ends of the slide with the tape or you will find your fingers stick to it and jerk the slide around when you are working.
b. You can usually fit about 15 pupae on one slide, so make two slides for each dsRNA you want to inject.
3) Collect pupae from jars about 5 weeks old at 30oC.
a. Note that the pupae will wiggle when you pick them up.
b. Try for pupae that are older – darker wings and eyes. They survive better.
c. Collect about 50-60 pupae for each dsRNA you plan to inject. That will give you 20-30 females.
d. Sort females from males (see http://www.ars.usda.gov/Research/docs.htm?docid=12892). Put 6-8 male pupae in the middle of the flour in each petri dish to mate with your injected females.
4) Collect your dsRNA at an appropriate concentration (1-4 ug/ul) in injection buffer plus dye, and keep it on ice. 5ul of RNA is enough to inject 15-20 pupae.
5) Place the female pupae on the tape – ventral side up. Stick the very posterior of the pupae to the tape. Attach as little of the posterior end as is feasible (usually only one segment), or they will not eclose properly. I find using forceps to position them on the bit of slide that isn’t covered with tape and using a blunted needle to push on the abdomen works well. They can be stationed fairly close together.
6) Take the following with you to the injection room.
i. a 1-10 ul pipet,
ii. a needle (or 2) for each dsRNA you want to inject
iii. your plates with flour
iv. the slides with pupae
v. RNA on ice
7) In the injection room
a. Turn on the nitrogen
b. Turn on the microscope light
c. Turn on the pump
d. Using the 1-10 pipet and a loading tip (extra long and slim) load a needle
i. Push the tip FIRMLY onto the pipet
ii. Suck up RNA (Be patient. It’s a little viscous.)
iii. Push the end of the pipet tip to the bottom of the needle
iv. Push slowly to eject the RNA. Move the end of the pipet tip out of the solution carefully so as not to introduce bubbles.
e. Place the needle in the injection apparatus
i. Raise the apparatus before inserting the needle
ii. Insert until you can see the back end of the needle, then tighten.
f. Open the needle (Note - we now have a beveller, so you can open the needles using the beveller before injecting.)
i. Put one of the slides on the stage near the needle.
ii. Lower the needle down until it is below the top edge of the slide.
iii. Push on the foot pedal until the RNA is near the tip.
iv. Move the needle carefully in and rub the tip gently along the edge of the slide.
v. Push on the foot pedal. If nothing comes out, rub the tip again. Repeat until a drop comes out when you press the foot pedal (Note – a small drop is nice because you do less damage to the pupa when injecting, but the needle will probably clog during the course of the injections. Just repeat the steps here when it does, then go back to injecting.)
8) To inject
a. Raise the needle up above the slide.
b. Move the slide in until the needle is above the first pupa
c. Lower the needle until it touches the pupa
i. Be ready for the pupa to move.
ii. Aim just to the right or left of the midline at the join between the 3rd and 4th abdominal segments (about where the wing tips point).
iii. Slide the needle quickly in, then pull back just a little before pushing the foot pedal. If nothing comes out, slide the needle back and forth a couple of times and try again. If still nothing comes out, re-open the needle.
iv. Slide the needle out and raise it a bit before moving to the next pupa
9) When all pupa that can be are injected, remove the ones that didn’t get injected. Place the slide with the injected pupa face down into the flour. Keep in the 30oC incubator (be sure there is a beaker of water in the incubator too.).