Dear all,
I am planning to isolate neutrophils from whole blood of pigs for later RNAseq analysis. The samples may be taken over the course of several hours, so I assume I will have to stabilize RNA to get the transcriptomic state at the moment of sampling.
My plan so far is to add RNAlater to the blood immediately after sampling. Neutrophils will then be isolated via Lymphoprep gradient centrifugation and the neutrophils suspended in RNAlater prior to RNA extraction.
Potential concerns:
- when freezing whole blood stabilized with RNAlater, the sample tends to clot. Would this be a concern over the course of 1-2h at RT?
- does the Lymphoprep density gradient still work when RNAlater is added?
- any other concerns or alternative ideas?
Thanks a lot!
Hello Bjorn Klabunde,
RNAlater is not recommended for preserving RNA in whole blood, plasma, or sera. Because of their high protein content, these fluids will form an insoluble precipitate if they are mixed with RNAlater.
Instead, you may use special tubes that contain a proprietary reagent for stabilization of intracellular RNA immediately upon collection. Intracellular RNA is stabilized for up to three days at room temperature. These tubes may be used for collection, storage, and transportation of blood and stabilization of intracellular RNA with subsequent isolation and purification of intracellular RNA from whole blood for further analysis.
You may want to refer to the links below for more information on these specialised tubes.
https://alitheagenomics.com/blog/how-to-preserve-rna-quality-in-whole-blood-samples#:~:text=To%20limit%20RNA%20degradation%2C%20blood,RNA%20shield%20blood%20RNA%20tubes.
https://zymoresearch.eu/products/dna-rna-shield-blood-collection-tube?srsltid=AfmBOoqFdHy9HnK-ShBusNKzGk57ubOLKxhtoY9qXNM_K0HgDYru9SQf
https://www.bdbiosciences.com/en-eu/products/blood-collection/blood-collection-tubes/paxgene-blood-rna-tube.762165
https://www.streck.com/products/stabilization/rna-complete-bct/
https://www.thermofisher.com/order/catalog/product/4342792
I am also attaching a few references that may help.
https://pmc.ncbi.nlm.nih.gov/articles/PMC4245490/
https://aacrjournals.org/cancerres/article/80/16_Supplement/759/644582/Abstract-759-RNA-Complete-BCT-A-novel-blood
https://www.sciencedirect.com/science/article/pii/S000991201200433X
Best.
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