I am having trouble detecting RNA viruses from whole blood samples collected under field conditions (lack of a real cold chain) from rodents that were placed in RNAlater. The samples were NOT separated first, no serum or white cells were removed, and I believe they were all collected in EDTA-coated tubes. I have tried spinning down the samples, removing the RNAlater and extracting RNA from the "pellet" of mostly red cells using a Qiagen kit (and similar silica membrane based technology) with little to no recovery. The down stream application is nested RT-PCR and sequencing of resultant amplicons. Any suggestions would be tremendously helpful!!!!
We've done this a fair bit in a variety of species for similar reasons as you are using it. Usually we just treat it as though it was whole blood and use qiagen or machery nagel blood kits (though I usually specify that the collectors use a ratio of 2:3 blood/supernatent to RNA later as that has worked ok for us in the past) , if you are only extracting from the cell pellet you will be loosing any virus that is in the supernatent. as someone above said haem etc in blood and the Mg binding effects EDTA (that is how it works as an anticoagulant, it binds heavy metal ions - its used therapeutically in lead poisoning for this reason) can really inhibit PCR - playing around with your magnesium concentration in your PCR can help with this. We've also generally found that qPCR is up to 100x more sensitive (and less contamination prone) than nested PCR but that won't help with your sequencing. The qiagen viral RNA kits are very good with samples that have large volume/low concentration of virus (especially if you add the carrier RNA - it really enhances nucleic acid recover). Have you checked that you are getting any RNA at all? (eg using beta-actin or "barcoding" primers). Of course the other possibility is that the rodents don't have the virus you are looking at, PCR positivity/circulating virus will be detected at an order of magnitude lower rates than seropositivity. For the sort of feild situation you are in we tend to use whatmans FTA cards for sample preservation and transport, which are fantastic as they are very simple and once dried can just be stuck on a shelf, though admittedly I've never tried to recover RNA off them.
Good luck!
There is a product you may want to check out here:
http://www.lifetechnologies.com/us/en/home/references/ambion-tech-support/rna-isolation/tech-notes/new-maximize-viral-rna-yield-from-biological-fluid.html
Be sure to keep track of how much EDTA is actually in your samples (solvated from the inside surface of the blood collection tubes). 0.15% EDTA (the usual goal of e.g. 3 mL of blood in a 3 mL blood collection tube) is about 3.7 mM EDTA ,,, just be sure you aren't incurring a problem from EDTA. Many RT-PCR enzymes are heavily inhibited by small amounts of blood components (heme, porphyrin, calcium, IgG, transferrin) so, if your downstream stuff isn't working well - it is likely the undesirable blood components you've carried along the way by trying to extract the RNA with red blood cells present. The Taq enzyme "Hot-Tub" has been reported to be the least inhibited by blood components - but may not be available in a format you want. Check the web for RT-PCR procedures/kits which can withstand RBC contamination. When in doubt, RNAzol and/or TRIzol always seem to succeed with every sample type for RNA yield purposes.
Have you tested put your blood samples directly in some extracting buffer i.e.AVL? We extracted arbovirus RNA from whole blood inthat way without RNAlater. We also use Avl for samples transportation.
Good luck!!!
Hello Katherine,
Please see: http://onlinelibrary.wiley.com/doi/10.1002/elps.201300190/pdf
Hi Katehrine,
I agree with Jack. Isolation is a kez step. We use QIAGEN viral or blood isolation mini kits and they work well.
There can be also a problem with sample storage. Try to store your viruses in RNAlatter on table as long as you stored them after the field isolation and you will see.
Good luck!
Jiri
We've done this a fair bit in a variety of species for similar reasons as you are using it. Usually we just treat it as though it was whole blood and use qiagen or machery nagel blood kits (though I usually specify that the collectors use a ratio of 2:3 blood/supernatent to RNA later as that has worked ok for us in the past) , if you are only extracting from the cell pellet you will be loosing any virus that is in the supernatent. as someone above said haem etc in blood and the Mg binding effects EDTA (that is how it works as an anticoagulant, it binds heavy metal ions - its used therapeutically in lead poisoning for this reason) can really inhibit PCR - playing around with your magnesium concentration in your PCR can help with this. We've also generally found that qPCR is up to 100x more sensitive (and less contamination prone) than nested PCR but that won't help with your sequencing. The qiagen viral RNA kits are very good with samples that have large volume/low concentration of virus (especially if you add the carrier RNA - it really enhances nucleic acid recover). Have you checked that you are getting any RNA at all? (eg using beta-actin or "barcoding" primers). Of course the other possibility is that the rodents don't have the virus you are looking at, PCR positivity/circulating virus will be detected at an order of magnitude lower rates than seropositivity. For the sort of feild situation you are in we tend to use whatmans FTA cards for sample preservation and transport, which are fantastic as they are very simple and once dried can just be stuck on a shelf, though admittedly I've never tried to recover RNA off them.
Good luck!
Hi Katehrine,
maybe it's best if you avoid ammonium sulfate containing solutions, such as RNAlater, for the vRNA isolated from whole blood all. Do you use indeed no solid tissue and the protein content in whole blood is also very high. Also this tissue is not as rich as RNases like tissue from the pancreas. As long as the viral RNA is bound in viral particles,the reduction due to degradation of plasma vRNA (extracellular virus) or leukocytes (intracellular virus) in EDTA blood is not so significant. The viability of leukocytes is guaranteed at room temperature in EDTA blood for several days. You could use instead a Ficoll-Paque density gradient centrifugation to perform plasma and WBC isolation. This will remove the erythrocytes automatically.
Good Luck!
Bernds answer is also a good point - I've treated supernatents wtih RNAases to get rid of contamination from lysed cells and then done viral extraction - the capsids are tough. If the samples are able to be transported in a few days Ficol would be a good alternative (though I'd imagine you only have very tiny amounts of blood unless the animals have been exanguinated). For the really appalling transport chain (6 weeks in 40 degree heat was one of our more noteable ones) however this won't work.
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