Can anybody please guide me as to how to isolate plasma for analysis of circulating microRNAs? I would like to isolate plasma immediately after blood collection (with high quality).
-Use an anticoagulant that doesn't interfere with PCR. See work of D. Duelli among others.
-Spin at low speed (600-800 x g range) to remove nucleated cells.
-Spin at high speed (2500 x g) twice to remove platelets. If this is not done, you will be measuring mostly platelet RNA, not extracellular RNA (consult work of M. Tewari and C. Pritchard; for more information, see recommendations of International Society for Thrombosis and Hemostasis and International Society for Extracellular Vesicles).
-For RNA extraction, use a biofluids appropriate method. We found that the Exiqon Biofluids kit works well, but others are also good options: https://www.researchgate.net/publication/236968834
Article Comparison of Methods for miRNA Extraction from Plasma and Q...
-Use an anticoagulant that doesn't interfere with PCR. See work of D. Duelli among others.
-Spin at low speed (600-800 x g range) to remove nucleated cells.
-Spin at high speed (2500 x g) twice to remove platelets. If this is not done, you will be measuring mostly platelet RNA, not extracellular RNA (consult work of M. Tewari and C. Pritchard; for more information, see recommendations of International Society for Thrombosis and Hemostasis and International Society for Extracellular Vesicles).
-For RNA extraction, use a biofluids appropriate method. We found that the Exiqon Biofluids kit works well, but others are also good options: https://www.researchgate.net/publication/236968834
Article Comparison of Methods for miRNA Extraction from Plasma and Q...
Daer Reyhaneh,
In our hemostasis lab, we isolate plasma from leuco , RBC and platelets for dosage of coagulation factors. We use anticoagulant tube with sodium citrate (commercialized; see on manufacturer websites: blue ones). We centrifugate whole blood at 3000 rpm (2500g) for 15 min at 20°C.
But your demand is particular, as you want, I suppose, nucleic acid (RNA), extracellular ones only, so in suspension in plasma.
We perfom also mutation diagnosis (coagulation factor V and II gene), but on DNA extracted from whole blood, and in this case, we ask to the physicians one EDTA anticoagulant tubes , which would be better for DNA analysis. But if they send us citrate tube by error, we can do DNA analysis (but on cellular DNA), and we do the same centrifugation and replace plasma + citrate by NaCl 9/1000, but because we use cellular pellet and determine SNP on leucocyte DNA.
But you, you want to conserve the plasma, remove the pellet and determine RNA on it; that is another problem.
As Kenneth said, we follow the recommendations from our thrombosis society, and there is few contamination by cells and platelets at this speed.
So, It is possible to perform genomic analysis on citrate tube, but perhaps that's less efficient than on EDTA tube, recommended (perturbation of PCR ? or reverse transcriptase ?).
But to my mind, the main problem would be the quantity of extracellular RNA existing in plasma (do you have an estimation ?), and the efficiency of extraction, whatever the technic used. So, I suppose you have optimized one to obtain the required extracted quantity for your further RNA analysis (sequencing, PCR, transcriptome ?).
Best regards
Didier
PS
Finally, thinking again to your problem and what we perform at the lab, you could do a "mix" of the two:
- as DNA anaysis is made on EDTA tube, you sample your blood on EDTA K3 tube,
- then, you follow the centrifugation protocol we use for coagulation factor dosages; and as that's not these proteins you want, but nucleic acid, nethermind: you obtain a plasma with EDTA.
But always, for me, the problem is the quantity of circulating micro RNA, which would be very low. But perhaps am I wrong ?
Didier
Usually the techniques to either isolate, detect, and/or characterize extracellular miRNAs may vary significantly from study to study... So you may need some trial and errors...
Here are a couple of options:
http://www.clinchem.org/content/57/6/833.long
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3167594/
This here may also be a little more useful as it contains a step by step:
http://www.exiqon.com/ls/Documents/Scientific/microRNA-serum-plasma-guidelines.pdf
Hope it helps. All the best.
Have you tried the commercially available Plasma Prep tube (PPT) from Becton Dickinson? They work great and minimize preanalytical errors associated with molecular diagnostic tests
https://www.bd.com/vacutainer/products/molecular/ppt/
I agree with Kenneth regarding multiple centrifugations. This is a must. I also agree with Fernando's comment about variation between studies. If different processing techniques are used with different isolation kits and different detection methodologies, how can studies even be compared to each other? Another critical issue to think about before you start your studies is which miRNA will be used to normalize your results? Spike-in RNAs seem to work well as endogenous miRNAs vary between patients.
We use kits from Norgen specifically made for RNA isolation from plasma/serum. We've gotten good results from the kits. We use CPT tubes (sodium citrate) as we also isolate the PBMC pellet. I suggest that you keep the blood tubes at room temperature (cold activates platelets and may result in RNA release) prior to and during centrifugation. Once RNA is eluted from the beads/slurry/columns, we've had better luck concentrating the eluate using Amicon's Ultra YM-3 columns (versus ethanol precipitation). Our RNA yield was higher and more consistent.
Dear colleagues
Thank you all for your answers. I'll try your suggestions!
Best regards
We are happy users of Qiagen's serum/plasma miRNeasy micro kit. RNA concentrations are typically 20 ng/µl or higher in a 14 µl elution volume. At Biogazelle, we do RT-qPCR and small RNA sequencing on such RNA samples to determine microRNA gene expression patterns.
http://www.biogazelle.com/lab-services/applications/mirna
We use potassium oxalate/sodium fluoride-coated vacutainers, as suggested in Kim et al. I really like that paper, they took the time to compare different additives (figure 1 in the paper). I then extract with the miRNeasy kit from Qiagen.
Article Plasma Components Affect Accuracy of Circulating Cancer-Rela...
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