I will be setting up an experiment to assay gene expression in the brains (effectively heads) of my wasps (body length of wasp: head to abdomen c. 2mm). Due to the large volume of tissue required to get enough RNA this means very high replicate numbers. High reps means working very quickly at harvest stage to ensure consistent expression patterns across reps.
To cut to the chase....This means I do not have time to separate heads from bodies at the time of harvest. I am wondering if I simply throw the wasps into RNAlater will it diffuse effectively through the exoskeleton/head capsule to protect the RNA in the tissues in the head? And I could sort out heads/bodies later. Or would it be better to flash-freeze and use RNAlater-ICE during the thaw? I have the same concern with this.
Any thoughts from anyone with experience of this would be very much appreciated.
Nicki
Very Interesting question, similar to my case. Could you check one of the spesial journal in .... (lets me check first....)
Hi Nicola, did it work? I can see in the case of Robert they were looking for DNA, but I'm wondering whether the RNAlater would diffuse rapidly enough through the cuticle to preserve the RNA which degrades faster?
Hi Pablo, that was my thoughts exactly. It did seem to work though - we got very good quality RNA that was not degraded. Bioanalyser traces were excellent and the samples pass the QC requirements for RNAseq.
Hi Nicola, I am facing a similar experiment set up. I was wondering if you threw the wasps while they were alive to RNAlater? I'm having a hard time getting alive insects on each tube and was wondering about that. How much volume were you using per tube? how many insects were you adding to a tube? I'm using 200ul in an eppendorf tube per insect, which are similar in size to yours (circa 2mm).
Best regards,
Gabriel
can i use RNA later for homogenization of tissue before RNA extraction
It is recommended that you get rid of the RNA later before tissue homogenization.
Ok can i add it after homogenization and how will i remove it during extraction.
Thanks
It seems I didn't understand your protocol. Usually you would preserve your tissue in RNA later. Then you would remove the RNA later and homogenize the tissue. If you are able to homogenize the tissue immediately after collection then you probably would not need RNA later?
Hi Pablo German and Nicola Cook
Did you place the entire insect into the tube with RNAlater? Or did you cut it into pieces or squeeze them with a pestle? Thanks!
Hi Daniel Fenando Paulo ,
I was working with Varroa mites which are 1.6mm in size, so I was putting many mites in a 1.5ml tube with RNA later. If you are working with bigger insects, you may need to cut them or use a bigger tube. If you cut them or squeeze them I guess you have to make sure not to damage the tissue you want to extract RNA from afterwards.
Thanks for the reply Pablo German
I will work with fruit flies (Family Tephritidae, not drosophila), which are more or less "big" insects (adult = 6 to 8 mm long). I just want the RNA for RT-qPCR, and will use the entire fly for that, so no tissue-specific. I was thinking about squeeze them gently (once or twice) in 1 mL of RNAlater.
Daniel Fenando Paulo that should work. They are still reasonably small and you should be able to get quite a few into a tube.
- Badges
- Science topic
- Similar topics
- Biological Science
- Anatomy
- Tissue