Hello!
I am trying to detect RNA transcripts in C. elegans and I am wondering if anyone has tried RNAscope? If you have any experience optimizing the technology in worms, or if you have any general insight into the feasibility etc, I would really appreciate it.
Thank you!
Hello cfujii,
Yes, we have experience optimizing it in parasitic worms (Redirecting). Please contact me at jebaj@vet.k-state.edu. Thanks!
Hi cfujii,
I’ve tried Basescope. On FFPE slices it worked as advertised. However I didn’t get signal on whole mount worms, which is necessary for my application. I only tried small variations on the standard protocol.
In the meantime I got good preliminary results with HCR using their C. elegans protocol on whole worms (there is also a couple of published papers that used HCR in worms).
I haven’t tried RNAscope, which should be more robust than Basescope.
Hi AlexisW,
Thank you so much for your response!
If you do FFPE, I assume you slice the worms and place the sliced sections onto a slide? Do the sections stay stuck on the slides after going through the many wash steps? I am trying to figure out if there is a way to secure the specimen onto the slide, because my specimen often falls off the slides during the wash steps.
Thanks again for your input!
Yes, I prepared a large amount of worms, pelleted, added NBF, pre-embedded in LMP agarose, and collected this ~0.5 mL high-worm-density gel. I had our core perform the paraffin embedding and slicing; they collected the slices on “X-tra” slides. Each slide has a large section (about 2 cm x 1 cm) of paraffin, with smaller inclusions of gel (about 0.5 cm diameter typically). While the paraffin melted and washed away in the initial 60°C/xylene/EtOH step, the samples stayed on the slide.
When I attempted whole-mount individual worms on Superfrost Plus slides or on poly-lysine coated slides, they didn’t stick through the peroxidase treatment. I haven’t found a work-around.
Hi AlexisW,
That makes sense and thank you for describing how you do your FFPE! By any chance, do you have any insight into the feasibility of the cryosectioning as opposed to FFPE? Is FFPE typically better for worm samples compared to cryosectioning?
Thank you again for your help!
No idea, I have very limited experience with these methods.
Jeba’s paper also seems to use FFPE samples, they might be more experienced with that type of approach.
OK, thank you for letting me know!