Has anyone developed a good protocol for stabilization and subsequent isolation of miRNA from plasma/serum (not whole blood). PAXgene blood lyse cells so it will release intracellular RNA. RNAlater keeps cells intact but precipitates serum proteins that entrap cells and may do so with miRNA carriers. Probably the only solution would be separation of plasma from anti-coagulated whole blood and than preserving plasma. This raises another question - is stabilized plasma (with high content of RNAlater or PAXgene reagent) compatible with TRI/Trizol LS-liquid sample reagent?
Does anyone have any experience with this?
You're thinking about this the right way: you want to eliminate whole cells and platelets from your biofluid before storing and/or RNA extraction...without removing extracellular miRNA carriers. Here's a little comparison of methods that my lab recently did (you can see it on my profile, too).
http://www.ncbi.nlm.nih.gov/pubmed/23720669
The drawback to most kits, of course, is that many companies will not share information about reagents...even with researchers who have no financial conflicts and are giving good publicity to the companies...it's an unfortunate state of affairs in science whenever there's a part of our process we can't fully understand!
You're thinking about this the right way: you want to eliminate whole cells and platelets from your biofluid before storing and/or RNA extraction...without removing extracellular miRNA carriers. Here's a little comparison of methods that my lab recently did (you can see it on my profile, too).
http://www.ncbi.nlm.nih.gov/pubmed/23720669
The drawback to most kits, of course, is that many companies will not share information about reagents...even with researchers who have no financial conflicts and are giving good publicity to the companies...it's an unfortunate state of affairs in science whenever there's a part of our process we can't fully understand!
I am also interested in this so thanks for pointing out your article. We use the QIAamp Circulating Nucleic Acid kit to isolate cfDNA, but I have interest in optimizing for miRNAs in the future.
I got reply from Life Technologies' Technical Application Specialist that "...with serum we would not recommend using RNAlater. We have developed a protocol to store blood sample in RNAlater. However, this does not include plasma fraction alone. Further details regarding this protocol can be found on the following link, http://www.lifetechnologies.com/de/de/home/references/protocols/nucleic-acid-purification-and-analysis/rna-protocol/blood-fractionation-protocol-for-collection-of-white-blood-cells.html". From previous correspondence it seems that RNAlater in the sample could be contributing to a poor quality RNA characterised with high levels of salt. It was in context of using Trizol LS Reagent for RNA isolation.
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Biomolecules
- Nucleic Acids
- RNA
- Small RNA
- microRNA