RNAscope (In situ hybridization) to study gene expression in pain-related tissues


#21

Hi @achamess, I’m new to RNAscope and am interested in trying your methods for DRGs! I had a couple of questions on details…

  • Do you perfuse with saline or PBS prior to PFA, and does the rate of perfusion matter?
  • Could I substitute PFA with NBF? (There’s a giant jug in the lab but I heard it can go bad over time, even with the methanol…?)
  • For sample pretreatment, do you skip the hydrogen peroxide step prior to protease IV when using the v2 kit?
  • It seems like the fluorescein TSA didn’t work out well for you earlier… would you then recommend Cy3 and Cy5 if co-staining? How would you decide which gene to put in which channel?
  • Is there anything to be particularly careful of in terms of avoiding RNases and RNA degradation during any of the steps?
  • Finally, have you tried doing RNAscope in transgenic lines that express endogenous fluorescent proteins? We have a tdTomato line I’d love to use but I’m not sure if the protein will survive the RNAscope procedure.
    I don’t have much experience with ISH or microscopy in general so any tips and advice would be much appreciated! Thanks so much for your help!

#22

@june

  • Do you perfuse with saline or PBS prior to PFA, and does the rate of perfusion matter?

Sometimes yes. It doesn’t hurt. But these days, I just do immersion fixation (drop the DRG right in PFA).

  • Could I substitute PFA with NBF? (There’s a giant jug in the lab but I heard it can go bad over time, even with the methanol…?)

I make fresh PFA. It’s easy enough. RNAscope recommends NBF, so I bet it works. But I use PFA.

  • For sample pretreatment, do you skip the hydrogen peroxide step prior to protease IV when using the v2 kit?

I’ll check with my colleague.

  • It seems like the fluorescein TSA didn’t work out well for you earlier… would you then recommend Cy3 and Cy5 if co-staining? How would you decide which gene to put in which channel?

We ordered the Opal 520 and that took care of the problem. http://www.perkinelmer.com/product/opal-520-reagent-pack-fp1487001kt

  • Is there anything to be particularly careful of in terms of avoiding RNases and RNA degradation during any of the steps?

We don’t do anything special. Use recently procured tissue (<2 weeks is best in our hands). If you want, you can use RNase free reagents like MoBio grade EtOH and PBS. I do just because I’m superstitious but my colleague doesn’t and it works well for her.

  • Finally, have you tried doing RNAscope in transgenic lines that express endogenous fluorescent proteins? We have a tdTomato line I’d love to use but I’m not sure if the protein will survive the RNAscope procedure.
    I don’t have much experience with ISH or microscopy in general so any tips and advice would be much appreciated! Thanks so much for your help!

Good question. Yes. We routinely do this with both GFP and tdTomato. You have to recover the fluorescence with antibody because, as you suspected, RNAscope destroys the native fluorescence.

For GFP: https://www.abcam.com/gfp-antibody-ab13970.html (1:1000)
For tdTomato: https://rockland-inc.com/Product.aspx?id=40760

Good luck! Please let us know how your experiments go.


#23

Great, thank you! Super helpful!


#24

I am a colleague of achamess and adding some information about sample pretreatment.

We use both hydrogen peroxide (10min@RT) and proteaseIV (30min@RT) as pretreatment for the v2 kit.

Good luck:grinning:


#25

Thanks for chiming in @MGM


#26

Thank you @MGM and @achamess! I am interested in costaining for my GOI and nociceptor markers (Trpv1 etc) to quantify the extent of colocalization. I know my GOI is expressed in the DRG from qPCR but have no sense of whether it’s a high or low expressor. Would you still recommend the v2 kit for this, or try out v1 first? In any case, I assume putting the nociceptor marker in green and my GOI in red or far-red would be best…?


#27

Costaining with IHC or another RNAscope probe? I’d use Cy3 for Trpv1 and Cy5 for your GOI because we’ve found that has the lowest autofluorescence. Cy3 is brighter but it may be hard to find real signal if your gene is a low expresser. You can get a sense of the expression of your gene from gene expression databases or comparing your qPCR for GOI to another gene whose expression you have an impression about. We like the V2 kit. I’d use that.


#28

Sounds good, we will try that. I can do either IHC or another RNAscope probe–would you recommend one way over another?


#29

Do another RNAscope probe. IHC after ISH is tricky. Sometimes it works, sometimes it doesn’t.


#30

Got it! Excited to try this out.


#31

Hello again! I was curious whether section thickness makes a big difference in your hands?


#32

We do 12-14 um. I wouldn’t do more than that.


#33

Would thinner sections hurt staining, say 7 - 10 um?


#34

If you can slice them, go for it.


#35

I had a chance to try out the single color chromogenic assay with ACD’s positive and negative control probes, and that worked out well! DRGs were fixed but went through the fresh frozen pretreatment like you recommended. Makes me more interested in their duplex chromogenic kit if I just need two colors, do you have any experience with that? In any case, thanks for all the tips!! Saved me a ton of time!!


#36

Great. Yes see the thread posts. That’s all we use. Works just as well. Good luck.


#37

Hi, we are starting some experiments using Rnascope in DRG and Spinal cord from rats. We will be using RNAscope® Multiplex Fluorescent Reagent Kit v2 Assay and TSA. We have fresh frozen and fixed frozen tissue (I collected the tissue and fixed in PFA 4% by immersion overnight), but we could also perfuse if that gets us better results. I took notes o the tips on the previous comments but I wanted to know if there any update on the suggestion (like perfusion vs just dropping the tissue in PFA) before we start! We will be looking at things that are low expressed.


#38

Thanks for joining the forum. I don’t have experience with rat tissue but I suspect the major difference between mouse and rat will be the size of the tissue. So I can’t speak to how well the PFA permeates the tissue. Mouse DRG is so small, i think it’s very fast. I notice no difference between perfused vs. drop fixed. You should try fresh, drop fixed, and perfused alongside each other on the same slide and see if you notice any differences. I suspect probably not. If you do the drop fixation though, I recommend not too long. In mouse, we do 2 hours max. It might be different for rat. if you fix too much, the signal is reduce. @tberta has experience with this.

So you’re going to have to systematically try stuff. The major parameters are:

  1. Fixed vs frozen
  2. Pretreatment: which protease, for how long, +/- antigen retrieval.

For fresh frozen, just follow the fresh frozen pretreat protocol as described by ACD.

For drop fixed, I think you should not just do ‘overnight’ but be precise about # of hours fixed. Try 2, 4, and 8 hours side by side using the fresh frozen protocol. we use the fresh frozen protocol even with lightly fixed tissue (with the omission of the 15 min PFA pretreat). You will likely see different signal intensities based on fixation time. We avoid the boiling antigen retrieval with DRG tissues because they are fragile. @MGM can speak more to this. Unless you have a strong reason for long fixation, I think the less the better.

Also, with the TSA kit, take note about the comment about Opal 520 vs FITC. If you’re going to do green, get the Opal.

If you’re looking for low expressed genes, use Cy3 or Cy5 for those. Probably Cy3, since it’s brightest. There is always the most autofluorescence in the green channel in DRG. So if you use green, use it on high expressed genes (like Actin or Nav1.7).


#39

Thanks for the response! I will update with results !
Marta


#40

@mvhamity How’d it go?