I have 0.5x0.5cm of rat colon sections frozen in RNAlater, the anatomy of the sections are barely discernible if at all and they are somewhat shrunken. Is it still possible to isolate the mucous layer from these samples? Also, there is a viscous opaque layer at the bottom of the tubes, could this be the mucous layer separated from the tissue? Any ideas would be very helpful.
Hi Nicolaas,
one method is to stabilize the tissue sample on a flat surface and scratch off the mucous layer with a microscopic objective glass. But it may be difficult regarding the size of your tissue samples. We have used it on fresh tissue, but it is beneficial for you that it is already in RNAlater sice this method crushes the tissue.
Dunno if this could help you, anyway good luck.
Hi Nicolaas Indeed, there is a chance, that the epithelial cells (mucosa layer) is separated from the rest of the tissue (connective tissue, muscles). What about to vigorously vortex the tube (or otherwise mechanically treat the tissue) and collect and extract RNA from whatever is coming off. Maybe you know a specifically expressed gene which kyou could detect by RT-PCR. In pig we also use glase slides to scratch the mucosa layer from fresh tissue before we put it in liquid nitrogen or RNAlater. Good luck
Hello Nicolaas,
Me and my colleauges also used microscpoic slides to scratch mucus from the fresh tissue of pig's stomach. Try to scratch gently form your tissue, however i think it will be very little of mucus from such small size of tissue. Be aware that mucus dries out fast when it is subjected to dry atmosphere. You have small size of tissue, dehydration will be very fast. Usually mucus contain approximately 95% of water. The relative humidity of air is much lower, so mucus will start to dry out very fast.
I believe that a viscous opaque layer at the bottom of the tubes could be a mucus layer which is loosely adherent mucus layer on colon. if you take a look on this reference "Atuma, C. et al. Am J Physiol Gastrointest Liver Physiol 280: G922-G929 2001", they provide with the thickness of gastric and colon mucus. Also Julia Davies from Malmö Högskola (SWeden) is the person who has experience in isolation of mucus, try to search fore her papers
Hope this answer will help you! Good-luck!
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