Hi Imke

Running microRNAs from serum, plasma or any body fluids is always challenging and in our opinion arrays aren't the best solution.

At NXT-Dx, we offer miRNA expression profiling using the latest in qPCR technology. We have compared qPCR,with arrays and sequencing techniques, especially in body fluids such as serum. The qPCR approach clearly comes forward as the optimal solution. On top of this we specialize in low-input approaches with this technique, so are very capable in delivering high quality data from serum samples. We can also take care of the RNA isolation from your serum samples before starting the

If you are still planning to run this experiment and would be interested in learning more about our approach, please send me an email on [email protected].

Maarten Braspenning

NXT-Dx

Hi Imke

Running microRNAs from serum or any body fluids is always challenging and in our opinion arrays aren't the best solution.

At NXT-Dx, we offer miRNA expression profiling using the latest in qPCR technology. We have compared qPCR,with arrays and sequencing techniques, especially in body fluids such as serum. The qPCR approach clearly comes forward as the optimal solution. On top of this we specialize in low-input approaches with this technique, so are very capable in delivering high quality data from serum samples. We can also take care of the RNA isolation from your serum samples before starting the

If you are still planning to run this experiment and would be interested in learning more about our approach, please send me an email on [email protected].

Maarten Braspenning

NXT-Dx

Thanks a lot for the offer, but this is a pilot study, so we do not have enough funding to outsource.

Dear Imke:

I am analysing circulating miRNA in human samples also, at the beginning of my work I compared serum versus plasma, and compared trizol extraction versus column based extraction methods, plasma samples gave better results in extraction and in subsequent miRNA analysis.

regarding the method of extraction it seems that column based method (Exiqon biofluid in my case) gives more total RNA with higher purity when compared to trizol method, however it is much more expensive.

best luck in your work

Dear Rana, thanks a lot for the useful information and the good wishes. When you go for plasma, is it more likely to get contamination from other RNA sources, than just miRNAs transported through the bloodstream with mircovesicles or bound to small peptides? Did you ever control for that? How did you do it?

Dear Imke:

the most important thing (for serum and plasma) is to avoid haemolysis as the rupture of RBCs will release its microRNAs to the circulation and affect your results. so we do a spectrophotometric analysis of the samples to detect hemolysis (absorbance at 414 with cut off level of 0.2). I also do a second centrifugation step to get rid of any cellular contamination in my sample (platelets or WBCs)

what speed do you use for centrifugation? I tried serum and used a protocol from a publication with 2x 300g for 10min each, but this seems to not work for me. I need at least 600g for 20min, but am afraid the more g the higher chances of hemolysis. Visual observation of my samples seems fine though, nothing purple visible, I did not do spectroscopy yet, but plan on doing it, too. Any tips?

Dear Imke

for plasma collection we centrifuge at 1300 g for 10 min at 4 degree (depending on the MMI guidelines for standardized biobanking). it works fine with me as I have a low rate of haemolysis

to reduce cellular contamination I do a second centrifugation of the plasma at 1940 g for 10 min. then store samples at -80 for further processing

Regards

Thanks a lot!