Total Exosome Isolation Reagent (from urine)

For the short-term, exosomes can be stored at 4 degrees C for up to 1 week. For the long-term, exosomes can be stored at -20 degrees C or -80 degrees C. When storing exosomes for the long term, it is important to consider whether they will need to be thawed more than once for the target application. If multiple applications (and thus multiple thaws) will be used for analysis, then we recommend aliquoting the exosome resuspensions into multiple tubes so that each tube will only undergo one freeze/thaw cycle. We have found that multiple freeze thaw cycles can cause damage to the exosomes and reduce their numbers.

Unlike serum, plasma contains numerous clotting factors and some additional proteins that can make it difficult to work with. We‘ve provided two protocol options, one with proteinase K (PK) and one without, in order to ease this difficulty. The protocol using PK is most useful when the end goal is analysis of the RNA or protein cargo contained inside the exosomes. It can also be used to isolate exosomes for use in other downstream applications, but it is most useful for RNA and protein analysis. The protocol without PK also isolates good quality exosomes, just not quite as pure as the PK protocol. The “no PK” protocol is more useful for isolating exosomes that will be used for surface protein analysis or electron microscopy identification.

There are several possible reasons why Western blotting analysis is challenging:

1. Not enough sample volume added. Exosomes can contain a fairly low amount of protein cargo, so for an initial experiment we recommend adding as much of the sample as possible.
2. Antibody concentration should be titrated. Also, they should ideally be used fresh and need to be stored properly.
3. Depending on the exosomal surface marker, certain gel conditions might be more optimal for the target antibody (e.g., reducing/nonreducing and denaturing/nondenaturing). We suggest checking with the manufacturer and exosome community about which Western blotting conditions are recommended for the specific marker you are targeting and the specific antibody you are using.
4. General Western techniques. Westerns can be tricky so we recommend the use of a positive control for initial testing to make sure the entire workflow is functioning as it should. Any protein or antibody can be used as long as they meet the conditions you need (e.g., denaturing vs. non-denaturing). In addition, when picking the protein, try to steer clear of those that are present at very high or very low concentrations in your sample to prevent overloading the blot or total absence of signal.

This can vary depending on the sample type, volume of sample, isolation method, and exosome content/concentration. Listed below are some examples:

1) When exosomes are isolated from 30 mL of HeLa cell culture medium using the Total Exosome Isolation Reagent, it is possible to recover approximately 8 ng exosomal RNA.
2) For exosomes recovered from 4 mL serum, approximately 2 ng exosomal RNA can be obtained.

In both cases, these amounts of RNA are sufficient for RNA library prep for Ion PGM or Ion Proton sequencing. For real-time PCR analysis, substantially smaller amounts of RNA are needed and much lower sample volumes can be used. For example, RNA recovered from 3 µL serum or 30 µL medium is enough for one qRT-PCR reaction.

No, the described effect does not have a negative impact on the RNA recovery.