In fact small RNAs and specially miRNAs are extremely stable in plasma. There a re a few studies reporting that even at room temperature during 2-3 days you have no significant decrease of the levels of some miRNAs in serum. The size of those molecules is an advantage in terms of stability because it conditions their degradation by nucleases.

In relation to the storage, I routinely store the samples at -80ºC, and in my hands they are able to pass by 3-4 freeze-thaw cycles without decrease in miRNA concentration. However, take a look at the enclosed document which is a guide by Exiqon about all those topics a few more.

In fact small RNAs and specially miRNAs are extremely stable in plasma. There a re a few studies reporting that even at room temperature during 2-3 days you have no significant decrease of the levels of some miRNAs in serum. The size of those molecules is an advantage in terms of stability because it conditions their degradation by nucleases.

In relation to the storage, I routinely store the samples at -80ºC, and in my hands they are able to pass by 3-4 freeze-thaw cycles without decrease in miRNA concentration. However, take a look at the enclosed document which is a guide by Exiqon about all those topics a few more.

By the way... I forgot... store always the samples separating the cells from the serun/plasma. Do not store the whole blood to profile circulating miRNAs. In this case you will get degradation.

I agree with Paco about miRNA stability and freeze thaw cycles. We too have observed no degradation after few freeze-thaw cycles. The only methods to recover Total RNA from whole blood after few days from collection is to use PAXGene Tubes from qiagen, but you cannot extract circulating miRNAs (or serum). So I would suggest to isolate serum first, than store at -80°C for further use.

Hi Mourad, miRNAs are indeed extremely stable in plasma and can be freeze/thawed multiple times (as others described above). We use TriZOL to isolate them from plasma. Just dilute the plasma in 5 volumes of TriZOL and follow the original trizol protocol as supplied by the manufacturer. Be careful not to use heparinated blood samples as this will influence the analysis. Always use citrate or EDTA samples.

Good luck!

Dear all, Many thanks for you all for the informative replies which covered several angle of my initially raised questions. I found it extremely useful especially the documentation provided by Francisco. Best regards. Mourad

Guido, do you use Trizol or Trizol LS?

Normally Tiazol LS is best suited for the isolation of RNA from biofluids. Both TRIzol and TRIzol LS are identical solutions except for the fact that LS is more concentrated which is advantageous to acomodate more starting sample materials, thus increasing the final yield. Hence, LS is very useful to use in extracting RNA from typical low yield samples (e.g. blood, plasma, serum, urine, etc....). You can use TRIzol LS for tissues for example but NOT before dilution.

@ Andrea. As Mourad says, we currently use TriZOL LS to isolate miRNA from plasma instead of the regular trizol. In the past, however, we used the regular trizol and that also worked fine.

I am using also Trizol LS, as Mourad pointed out, because is better for big volumes of starting material.

It was just to know if Trizol could be as good as LS...

We routinely use the miRNeasy kit.

Thank you Jo. It's a honor to exchange information with you. You developed a software that is a milestone for many researchers involved in miRNA quantification by qPCR! Thanks again. Andrea

Thank you for positive feedback!

@Jo: What software Jo?. Is it specific for miRNA RT-qPCR relative quantification of gene expression?. Please let me know whether is is publically available so I can try it. Thanks. Mourad

Dear Mourad,

if you freeze the whole blood sample, you will never be able to isolate plasma RNA from RNA deriving from leukocytes... I think that you have to centrifuge the plasma as first step.

freezing the whole blood leads to cellular RNA contamination as a result of WBCs lysis. so it is essential to process the entire blood samples for isolating the plasma quickly.

it is said that extracellular RNA are kind of protected; they are very stable and you don't need to use any reagent (like RNAlater) to preserve them while freezing. although several freezing and thawing processes could somehow decrease RNA level, but it is negligible.

We are currently storing the serum and plasma samples with 1 mL of qiazol ragent with good results.

@Guido: whan you used Trizol for isolation of miRNA in plasma, which amount of plasma did you have? I expect only 100uL (from mouse.. i know it's not the subject but..).

@Jo: when you say "We routinely use the miRNeasy kit." are your talking about miRNeasy mini kit for tissue/cells or miRNeasy Serum/Plasma kit?

To isolate extra-cellular mirna from blood serum and follicular fluid of bovine previously we have used miRNeasy mini kit which is actually for tissue/cells and it works nicely. Now for our new project we are using miRNeasy serum/plasma kit. Interestingly, I found that there is no significant difference in the concentration yielded by both kit.

For isolation of microRNAs from serum or plasma, we use miRNeasy serum/plasma micro kit. For isolation of total RNA from tissue or cells, we use miRNeasy mini kit. Indeed, serum or plasma need to be prepared prior to freezing the blood sample. If you are looking for gene expression analysis of cellular RNA from blood, then we recommend the PAXGene tubes and matching RNA extraction kits (there is one for total RNA and one for microRNA). The software that was reference to was Biogazelle's qbase+ qPCR data-analysis software.

http://www.biogazelle.com/qbaseplus

PAXGENE total RNA extraction kit system..it comes with its own blood collection tubes.. in contrast to other usual blood tubes..it has the RNA preservative in the collection tube ( to reduce any changes in the gene expression profile once the blood is out of patient).

Once blood is collected..the blood collection tube can be stored at 4 degrees for 7 days, or at -20 or -80 degrees freezer for weeks ( they say in manual for months before prcessing)...it preserves both mRNA and miRNA species both together...after collection and before processig it should be left at room temperature for 2 hours for incubation...it is better to avoid thawing and freezing for integrity mRNA...the kit however has been validated for differnt storage times and against different refence genes with success

http://onlinelibrary.wiley.com/doi/10.1002/jcla.20075/abstract;jsessionid=7E39792DC7FD3169F246ED7761FFC760.d02t04?deniedAccessCustomisedMessage=&userIsAuthenticated=false

My dear, I agree with Francisco, microRNAs are very stable and during separation plasma from blood cells, they are stable and after that you can keep it in -80C for long term without significant decrease. of course you use PAXgene blood miRNA kit too but  DNase, RNase-Free Reagents and Buffers; to be used in conjunction with PAXgene Blood RNA Tubes.

best

mehdi

Dear all,

Just asking for updating; from your latest experience, what is the best method to extract RNA (including miRNA) from plasma if it is needed for NGS RNA sequencing? do you use a kit for extracting only miRNA such as NucleoSpin® miRNA Plasma (MACHEREY-NAGEL)? or do you use a kit for extracting the total RNA such as miRNeasy Serum/Plasma Advanced Kit (QIAGEN)?

I am wondering if you use the total RNA for NGS sequencing, high proportion is mRNA, have you any idea/experience how that could affect to the sequencing of the small RNAs?

Thank you,

Best,

Salam

Can miRNA remain stable in plasma if stored at -4 Degrees ?