This is an interesting question and there is no definite answer, we can only explore together what loading controls are meant to achieve and how to apply them to the very specific case of isolated exosomes.

So if you extract whole cells and then measure a specific parameter, for instance a specific protein of interest that may be upregulated, downregulated or differentially modified (processed) in response of a certain variable (anything, time, temperature, drug, disease, mutant....), then we use often total protein as a loading control. Some people use an abundant house-keeping gene, like actin, but reviewers often ask if the house-keeping gene is really neutral, so total protein is usually the best because it averages out random variables. Essentially, the total protein sample controls for how well you extracted the cells (how much elbow grease you put intro grinding up the tissue in a mortar or how well you sonicated the cells). If you then load equal amounts of protein, any differences in your protein of interest may be attributed to the biological process you are studying with your variable.

If you are working with exosomes, you are working with a specific fraction of a more complex sample, and so you ask the question 1) how well did you isolate them, 2) what is the yield, and 3) what is the level of contamination (cell debris, extracellular fluids, membrane vesicles from disrupted organelles etc....). It all depends on how you have isolated the exosomes. For this you need markers (that are not expected to be present in exosomes) to check contaminants. When it comes to yield you need specific components that are targeted to exosomes, but if you want to study if certain proteins are targeted to exosomes under specific conditions, then it is essential to have a random component as control, something that is always present in exosomes. Exosomes contain cytosol from the cell they are derived from, and this is an aqueous phase with water-soluble components found in the cytosol. Certain mRNAs will be highly soluble in water and enter exosomes by unspecific bulk flow. In contrast, membrane proteins or specific ligands associated with membrane-spanning receptors may either be enriched or depleted in exosomes. Furthermore, detecting mRNAs is highly sensitive through PCR methods and can be achieved with much less material than detecting soluble proteins that may be present in the exosome lumen by unspecific bulk flow. Measuring total protein content of exosomes will often be below the detection limit. For this reason, I believe detecting abundant water soluble mRNAs as general cytosol marker is appropropriate to determine exosome yield. Ideally, one would measure several mRNAs. Controlling exosome purity is another matter of course.

Thank you Jürgen Denecke for the quick answer. I really appreciate the time and effort you put into your detailed answer. Thanks for your help.

Hi, really interesting reply from Jürgen Denecke.

Totally agree about why people rather look into RNAs rather than protein, yields of exosomes are always an issue haha.

In my experience, you will select a specific hosekeeping microRNA as internal control. Ideally, transcriptomic/RNA-sequencing data will reveal microRNA candidates that are no changed whitin your conditions (treatment/no treatment). Alternatively, there are few microRNAs, like let-7g, I think they have been demostrated to be a good housekeeping control for RNA in serum. Therefore, you could always validate them whitin your conditions and see if the Ct differ in your exosome samples across replicates and condition. This have the caveat that your qPCR skill are good enough to be loading the same amount of cDNA into the reaction.

I hope this has helped a bit :D

Thank you David Roig-Carles for your reply.

Yes soon we are shifting from mRNA to miRNA and that is indeed helpful.

However, my case is even more complex as we work on diabetic nephropathy and in diabetes, as you know, many so called " housekeeping "genes such as GAPDH are altered for example which makes the choice of an optimal loading control even more challenging.

Exosomes are such a novelty in biomarkers field therefore overcoming their challenges is a must.