Screwed up blood extraction/storage, am I screwed? - (Feb/02/2014 )

Hi,

I needed to extract serum from rat blood samples in order to measure melatonin later on using ELISA. However, after the blood was extracted from the rats, we did not let the blood sit at room temperature to allow coagulation before centrifuging to extract the serum. Instead, we put the blood samples on ice, and then centrifuged the samples (~40-60 mins later for the first samples & ~10-20 minutes for the last samples), extracted the supernatant, and then froze the extracted supernatant and what was left in the original tubes in L. Nitrogen and stored it at -80. So I have a couple of questions:

1. In terms of the "serum" that I extracted, will it still be accurate and useable for ELISA analysis even though we did not let the blood coagulate at room temperature before centrifuging. 

2. In terms of the blood/serum phases that were left over in the original tube that were frozen and stored at -80. I tried thawing some of the samples in a water bath to pipette the samples inorder to extract RNA, but the samples were frozen right after being centrifuged and so were still separated into two phases. As a result, only the top portion could be pippetted, which I am assuming is the "serum," and the bottom part was just chunky and could not be pippetted. Are these samples useless now for RNA extraction?

Thanks for your help.

David

What you have got is not serum - it is plasma (serum is plasma without the clotting factors and blood cells).  I don't know how this will affect any measurements by ELISA, but I suspect it won't affect them much.

Thanks for the response. You are right, it is probably plasma as ice slows down clotting. However, the normal protocol to get plasma is to add anticoagulant to ensure no clotting, I doubt the ice is as efficient. So even though I probably have plasma, is there such a thing as less "pure" plasma and the plasma that I have will have less clotting factors or blood cells? Thanks.

David