Full disclosure; I work on brain, so I'm not familiar with your specific sample type.

That said: The most important thing for integrity of RNA in tissue is to work as fast as possible and as cold as possible. The perfusion procedure usually has a lag of a few minutes between cessation of effective circulation (when the animal is profoundly anaesthetised and heart rate and respiration are very abnormal) and effective cooling of the tissue (once cardiac access is established and cold PBS is running.) During this time, the tissue you want to work on is undergoing hypoxia and oxidative stress, which can make endogenous RNases more active, and could potentially also result in transcriptional changes. For this reason, we never perfuse before collecting tissue when the aim of the experiment is to analyse gene expression - rapid dissection on ice is technically easier, faster, and likely better.

The exception to this is if the presence of blood in your tissue is going to interfere with your results. If it is important for your specific experiment that none of the RNA or protein you're analysing came from blood cells, you should perfuse. I prefer to accept the presence of blood as the natural state of the tissue, because in my opinion there IS a difference in the effectiveness of perfusion between different animals - possibly because I am not very good at this technique, but I guess there is some variation even if the perfusions are done by someone very proficient. I feel like it is more reasonable to assume that tissue dissected without perfusion contains the same amount of blood between biological replicates, versus tissue that was perfused where it might be quite different.

I'm much less experienced with protein work than RNA work but I think most proteins are much more stable and unlikely to be degraded by a few extra minutes taken to perfuse the animal - some post-translational modifications are very labile though.

Hope this helps :)

I have never worked with nucleic acids, but I have done some (immuno)-histochemistry. And at least in that type of work it is essential that the fixative reaches every cell as fast as possible, to prevent post-mortem changes. If you simply cut out a piece of tissue and throw it into fixative, the diffusion distance between the surface and the cells in the core is several mm even for small blocks, fixation time is hours. If you perfuse the rat with PBS and then fixative, the fixative has to cross only a few µm from the capillary to the cells, that is, fixation time is minutes.

In addition, some tissues cannot be prepared without damage until they have been hardened by fixative (sciatic nerve for demonstration of diabetic damage, for example).

The technique is quite simple, open the chest of a deeply anaesthetised rat and with a scissor cut a small hole at the very tip of the heart, that is, the left ventricle. Insert a blunted 18 G needle (smaller, if the rats are below 200 g) through the ventricle into the ascending aorta and clamp it there with a haemostat. Severe the vena cava so that fluid can leave the circulation and start perfusing with PBS at a pressure of 2 m water-column. Within a few min, the tissue (best seen at the liver) will turn from blood-red to pale brown. Be prepared for heavy twitching of the rat during that process, it is a purely local reaction to changes in ionic composition and has nothing to do with pain or a flight-or-fight response. Nevertheless, it is not pretty.

In your case, I'd use ice-cold PBS to bring the body temperature down ASAP. I don't know whether RNase inhibitors are membrane-permeable, if they are it would make sense to include them in the buffer.

Thank you Laura Leighton and Engelbert Buxbaum

I am planning to dissect mice and isolate spleen, liver tissue samples to be stored in RNA stabilizer in temperature( -20 ) for later analysis.

I agree with Laura Leighton - the faster you take out tissue and freeze it at -80, the better, because RNAses may affect the quality of your RNA and all downstream applications.

That said, I have also taken out tissue for RNA isolation followed by qPCR after saline perfusion to get rid of blood contaminants (I am working with dorsal root ganglia and dissecting them takes time regardless of perfusion). But as Laura mentioned there is variability in the quality of perfusion between animals.

I would suggest you have a look if your target RNA is expressed in blood cells and design your experiments based on that.