My lab performs terminal bleeds on a regular basis, although we have not studied miRNAs.  However, I believe the easiest and most reproducible method is orbital enucleation.  The mouse must be deeply anesthetized (no response on a forceful toe pinch) and then you use a fine pair of curved forceps to remove one eye.  This should be done forcibly and quickly to ensure the blood vessels rip open.  Generally at least 0.5ml can be collected from adult mice and older bigger mice can yield over 1ml.  We collect into different tubes, with and without anticoagulant , as required for the experiment.  Since the blood is not forced through a needle and syringe, it does help limit hemolysis.  If your collecting from a lot of mice, I have seen a little hemolysis because the process takes long.  You might try collecting and putting the tubes on ice, but I'm not sure about the effects on miRNA.

I have seen people skilled in cardiac puncture make it look easy, but I still prefer orbital enucleation for speed, simplicity and reproducibility.  Only time I have heard that it doesn't work is with some strains of mice that are prone to blindness associated with an underdeveloped circulatory system in the eye.

Thank you for your answer! It's really helpful.

Concerning stocking the samples during the process, I am just hesitating whether or not putting the samples on ice because I am not sure if the thermic shock can lead to hemolysis (even a very small amount of hemolysed blood cells can contaminate my samples).

What do you think?

Thank you!!

Hi Yara

Are you needing to collect plasma or serum?

If plasma, then the ratio of anti-coagulant:blood and mixing are important consideration.  Either too much or too little anti-coagulant can induce hemolysis; likewise, too much or insufficient mixing.  

In answer to your storage temperature question, if you are needing to prepare plasma, I would suggest stabilizing the temperature of the blood as quickly as possible to around 20 oC and centrifuge as soon as possible.  If that is not possible, you need to be mindful that colder temperature will induce cold-storage activation of platelets, which may affect your miRNA results.  I would not place the tubes directly in contact with ice, but rather shield them by placing the test-tube rack on a protective sheet above the ice or cold-brick.

Regards, Rosemary