I am new to the field of circulating nucleic acid detection. I would like to search for microRNA down/upregulation in blood samples from patients suffering from certain type of cancer. I have never tried to isolate and quantify extracellular micro-RNA. Have you tried it? Maybe there is a good kit you can recommend? Is the 30-5-5 min serum centrifugation necessary?
Filip, see this post: https://www.researchgate.net/post/Share_your_experience_about_extraction_of_miRNA_from_plasma_or_serum_What_is_the_best_protocol_pitfalls_and_tricks_you_can_discuss_in_public
Ewa
Hi Filip!!
See this article:
https://www.researchgate.net/publication/249648487_Utilization_of_paramagnetic_microparticles_for_automated_isolation_of_free_circulating_mRNA_as_a_new_tool_in_prostate_cancer_diagnostics
http://www.ncbi.nlm.nih.gov/pubmed/23857647
Article Utilization of paramagnetic microparticles for automated iso...
Hi Filip. We are using this kit http://www.mn-net.com/tabid/11246/default.aspx (nucleospin miRNA plasma) from Macheney-Nagel with very good results. We use plasma, not serum for microRNA purification.
The small amount of small RNA in plasma samples makes quantification in nanodrop unsatisfactory, and therefore, we use directly 4 µl of the eluted RNA for the RT.
For relative quantification we are using a endogenous reference control (miR-16). From non-degraded plasma samples you will obtain Ct for this microRNA ~20.
You can obtain similar results using this kit from EXIQON: miRCURY RNA Isolation Kit - Biofluids.
Good luck.
Ginés
Hi Filip. I use this one
http://www.qiagen.com/es/products/catalog/sample-technologies/rna-sample-technologies/dna-rna-protein/qiaamp-circulating-nucleic-acid-kit
Those are very helpful answers. Thank you Gines Luengo-Gil for including sample Ct values as that's what I am most concerned about. Ct of 20 is really surprising. How much blood do you collect and do you use any specific spin protocol for separating plasma?
Hi Filip,
you need as little as 300 µl of plasma. If you want more amount of RNA, you need only to increase proportionally the amount of buffers in the kit.
Here you have the workflow (page 2):
http://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/RNA%20and%20mRNA/UM_SmallRNADNAPlasma.pdf
2) If you want to try another method that works well, you can lyse the plasma with TRIzol LS and then purify RNA with this kit from ZYMO research (Direct-Zol):
http://www.zymoresearch.com/rna/direct-zol
3) For obtaining plasma, we centrifuge blood samples for 15' at 1000g, and then, place the plasma aliquoted in 1'5 mL tubes and freeze immediately at –80ºC.
Ginés
Hi Filip, you might have a check about our recent paper on circulating small RNAs. I think you might be also interested in cheking the newly discovered tsRNAs, along with miRNAs population.
http://jmcb.oxfordjournals.org/content/6/2/172.full
You can also download the PDF file with Suppl data within my researchgate webpage:
https://www.researchgate.net/publication/259493728_Identification_and_characterization_of_an_ancient_class_of_small_RNAs_enriched_in_serum_associating_with_active_infection
Article Identification and characterization of an ancient class of s...
Hi filip.
We used saliva instead of blood. For profiling we used taqman microrna cards...for verification we used taqman mirna assays..both are from applied biosystems...good luck
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