Dorsal horn RNA?

spinal_cord

#1

Does anyone have a good technique for getting RNA from the dorsal horn? I’d like to try to look at dorsal horn gene expression at a couple of spinal levels.

Previously, after removing the DRGs, we tried cutting the spinal cord at the vertebral levels that correlate to specific spinal cord levels, and then slicing that in half coronally with a scalpel to separate the dorsal half from the ventral half. However, since we are doing fresh dissection (not perfused), the cord is very soft by the time we attempt to cut it in half, and it is difficult to reliably divide the spinal cord.

Any advice on how to best dissect this would be extremely helpful!


#2

@runDRG

First, here is a video I made for spinal cord dissection:
http://forum.painresearcher.net/t/mouse-spinal-cord-and-drg-dissection-video-active-users-only/196/1?source_topic_id=236
If you can’t access this, send me a PM.

You want to move as fast as you can. So do the opposite. Take spinal cord first, then DRG. You need to remove the SC to access the DRG anyway, right?

I use spring vannas scisors to cut the lumbar cord into 4 quarters. If you want to take just dorsal, cut it in half axially (like slicing a sub sandwich long ways). This takes some finnesse, but it works. If you want to be more precise, you’d need to use a Vibratome, but that is much more involved and probably won’t change much.

Keep everything clean. I use RNase-free PBS to moisten the cord so the scissors don’t stick.

Have cold tubes ready to snap freeze your tissue in (dry ice).

For processing, there are a number of kits. I like the Direct-zol from Zymo or the Qiagen RNeasy kit.

You can take the DRGs after if you want, but if you want SC, prioritize that and get to it as fast as you can. No more than 5 minutes to freezing. You can perfuse with cold PBS, oxygenated PBS if you want to try to buy you some time, but that’s more work.


#3

Thanks so much for your help! It sounds like switching our order of dissecting and keeping lots of PBS on hand should clear up some of these issues. Rats’ erector spinae muscles are a bit bulkier than mice, so I hope I’m able to slice through them as quickly as you did with your mouse!


#4

Glad to help. If you upgrade the size of your tools, it should probably work. How do you break the vertebrae? rongeur or something stronger?

Have RNase-ZAP on hand to clean your tools too. Clean down your dissection area as well.

Good luck. Let me know if you have questions.


#5

We break our vertebrae with (conveniently named) bone cutters, as they’re too thick for the rongeur you use for your mice.

I tried out your technique with a rat, and I think this approach will be much better for our spinal cord dissections than what we had been doing before! However, there was a pretty significant amount of blood, which flooded the spinal canal and made it much harder to dissect out the cord and DRGs. Any idea what vessels may be the culprit, and how to best avoid them for a cleaner dissection?

Thanks again for your help


#6

Glad you found a better approach. Bleeding sometimes happens. If you want, you can perfuse your rat with cold RNase free PBS beforehand. People do that because blood is a significant contributor of native RNases. But that’s one more step to do. I would just accept the blood and rinse the tissue when you take it out.


#7

Also, keep that PBS on hand and you can rinse the blood away and gauze it up. That will help you see what you’re doing.


#8

@achamess @runDRG, I just saw this forum and the video. I have one advice. In fact, we have realize in the last years that before to start the spinal cord dissection it is better to perfuse the animals (eg. PBS or saline). In fact, without perfusion there is a lot of immune cells inside the blood vessels that could affect your results. So even for PCR, WB or FACS we are perfusing mice with (PBS or saline) before spinal cord or DRGs dissection.


#9

Hey @thicunha. I think this is a good point depending on your goals. I don’t like that there is one more manipulation to make before getting the tissue out, but if you want to be sure that you’re only looking at resident cells, then this is important. Thanks for the suggestion.


#10

Hi @achamess. I agree it is one more step in the process. The problem is that the leukocytes inside the vessels express a lot of immune mediators in high levels. So, when you are analyzing inflammatory mediators and you believe you are measuring in the spinal tissue, probably part of these mediators are in these immune cells. Please try to consider this suggestion because we had several diferent resuts when we start to perfuse the mice for PCR and WB.


#11

@thicunha Yes. I think you know that your marker of interest is exclusively neuronal, then perhaps it doesn’t matter if you perfuse. But if that’s not the case and you’re looking at glial/inflammatory markers, perfusion is probably critical, as you’ve said. I will definitely do this when/if I look for inflammatory things in the spinal cord.


#12

@achamess Great. thanks


#13

@achamess @thicunha Thank you both for your suggestions! Modifying our dissection technique and adding a PBS perfusion step has made dissecting simpler and really improved the quality of our samples.


#14

That’s awesome! I’m so happy. Thanks for sharing. This experience is exactly what this forum is for. Keep us posted.