Dear David,

We have tested EXIQON Exosome Isolation Kit for 1.4 mL plasma samples and then followed the "Appendix" section to get exosomal RNA, but we're having very low quantity of RNA (below 3 ng/μL). 

Are you following the entire exosome isolation protocol or have you tried some alterations in order to get a better yield?

Thanks for your attention,

David Bidarra

Hi David,

I am isolating from conditioned medium of cell culture, so probably my yield can't be compared with yours.

However, I follow all the steps in the isolation and I resuspend the pelleted exosomes in Quiazol (following miRneasy protocol).

My yield is about 25 ng/uL in a volume of 50uL for around 7 million cells (maybe with that you can think if your yield makes sense), but apart from the extraction method (which was chose just because we had it already in the lab) I havent altered the protocol.

I do between one hour and hour and a half of incubation with the precipitation buffer and seems to be fine but I havent tested if it improves if you leave it overnight ( as it said it can be done in the protocol).

BW,

David

David Sir- how much volume of cell conditioned media have you tested with the Exiqon Exosome Isolation kit.Ultracentrifugation protocol would not be feasible to be followed for mirna mimics experiment so it would be of great help if you can provide me the feedback for the Exiqon kit. Thanks

Hi Nisha,

I was working with cell culture supernatant so my volume media was quite high. In order to save reagent, I previously concentrated using AMICON filters. However, I run some comparisons with Ultracentrifugation. In terms of yield, Exiqon tended to yield a higher amount of exosomes than Ultracentrifugation. However, western blotting and protein quantification indicated that the purity of these exosomes was worse than ultracentrifugation one. So keep that in mind if you want to avoid extracellular protein to be carried in your experiments.

I think the lowest amount anyway of media I tested was around 10 mL (prior concentration to 1 mL) (equivalent to a FK of 75 cm2 of confluent cells), and the yield was in the limit of detection. So keep that in mind because depending on your cell line and conditions you might end up with no enough exosomes.

Anyway, if you have more question and need help, drop another message and we can have a thought

Good luck

David

David Sir- miRNA mimics experiment are carried out in 6 well plates or 24 well plates which already would have only 1 ml of media. So how to carry out Exosome isolation. I haven't found any article mentioning the exosome isolation strategies for the transfection experiment.If you can help me on this part with your experience it would help me in my research.Have you tried other kits as well?

Hi Nisha,

I think that amount of starting cells might not be enough to isolate a proper amount of exosomes for further experiments.

So I would recommend to change the experimental design.

I am planning to knockdown some microRNAs in my secreting cells to study their role in the exosomes. I would recommend to optimize a transfecting method on a larger cell surface ( at least one fk of 175cm). You can KO your gene of interest by using cheap transfecting reagents or decide to transfect the gene in a stable way using CRISPR or lentiviral transfection.

Unfortunately, these are the only approaches I can think of that are feasible and money-friendly.

Some people have reported you can transfect your exosome preparation once is already isolated. However, keep in mind that transfection might change your exosome completely and that can bias your results.

Good luck,

Davi

Hi Nisha,

Our exosome isolation kit works well with transfection or well based assay system. You could get up to a total of 1 microgram of exosome RNA from 10^6 cells in a well