Hi Alessandro,

My first point is, if you are interested in extracellular miRNAs, you must avoid whole blood as it contains cellular fractions. it is wise to use plasma or serum samples for extracellular miRNA study.

Second point is choice of fluid fraction. There are at least two major populations of miRNAs in plasma or serum samples- vesicle enwrapped or ago2-coupled. You can either use whole plasma/serum sample or fractions.

Third point is which isolation kit you are going to use? In my experience miRNeasy plasma/serum kit or mirvana kit is best for circulating miRNA isolation.

When we used whole plasma (500 ul), we got on an average 5-10 ng/ul (final dilution with 15 ul water at the end of isolation procedure). If you need higher concentrations just increase the input volume.

When we used exosomes from 10 ml plasma, we got around 15-25 ng/ul (same isolation procedure)

Hope this will help you.

"Full microRNA genome profiling is possible from

a total input of as little as 35 μL of serum/plasma

per plate" - source: http://www.gene-quantification.com/microRNA-serum-plasma-guidelines-exiqon.pdf

you can also check these articles:

http://www.pnas.org/content/105/30/10513.long

http://gene-quantification.net/mcdonalds-et-al-circulating-microrna-preanalytical-2011.pdf

http://www.sabiosciences.com/manuals/Whitepaper_Serumplasma.pdf

I think probably you will get your answer from these references.

Thanks

Hi Alessandro,

My first point is, if you are interested in extracellular miRNAs, you must avoid whole blood as it contains cellular fractions. it is wise to use plasma or serum samples for extracellular miRNA study.

Second point is choice of fluid fraction. There are at least two major populations of miRNAs in plasma or serum samples- vesicle enwrapped or ago2-coupled. You can either use whole plasma/serum sample or fractions.

Third point is which isolation kit you are going to use? In my experience miRNeasy plasma/serum kit or mirvana kit is best for circulating miRNA isolation.

When we used whole plasma (500 ul), we got on an average 5-10 ng/ul (final dilution with 15 ul water at the end of isolation procedure). If you need higher concentrations just increase the input volume.

When we used exosomes from 10 ml plasma, we got around 15-25 ng/ul (same isolation procedure)

Hope this will help you.

Maybe this paper can help you in addition.

Thank you all for your answers. All of them help me to understand if I'm moving towards a really applicable biosensing device. At now, I don't have any idea of the extraction protocol, because we are setting-up the assay with synthetic miRNA. In the very next future we will discuss with our colleagues for real samples and we will see which can be the best solution, probably serum/plasma.

Thank you again

hi .. i dont know if this will help you .. any way best of luck with your project 

i've extracted miRNA from plasma and serum and checked the concentration it was ranged from 3 to 4 ng / microl liter .. 

Hello,

If you are trying to extract "total circulating miRNAs" from serum and/or plasma, just be aware that these ranges differ. Not only are these dependant on physical activities, but also there is a chance of external/alien miRNAs surviving and expressing in our system.

We were amazed as well, and thought it is worthy of mentioning this as a hypothesis, which was recently published. If you have, kindly go through the same and while mimicing the natural miRNAs with synthetic ones, this plot needs to be kept in mind as well, please.

Good luck.

Warm regards,

Venk

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