Exosome isolation was performed from serum (Exosome precipitation reagent, İnvitrogen), and then when RNA was isolated from exosomes with a total RNA isolation kit(Magmax, miRVana) and measured with qubit, the amount of miRNA was found to be approximately 0.5-2 ng.
Is this amount of miRNA sufficient for cDNA reaction? Evaluation with internal control RNU48 no expression peak observed? What could be the reason for this?
I know that our core uses a bead-beating robot for serum exosome RNA isolation. All of the salt in the serum can be tricky to allow for a good isolation. That being said the amount you get out will likely have to be analyzed by BioAnalyzer. Exogenous spike-in miRs should be used to track extraction and normalize results.
Firstly, thanks for your response. We chose the isolation method from the serum with the kit method. Could you make any suggestions about exogenous control used in exosome studies? And why should exogenous control be used?
Hi Emine,
1. Please isolate exosomes from serum (stand alone) using Invitrogen™ Total Exosome Isolation Reagent (Catalog number: 4478360).
2. For quality check, you can perform anti-CD-63 immune-labeling (flow cytometry/confocal microscopy) to have an estimate of the exosomal population isolated.
3. Subsequently, you may isolate the miRs using Takara's NucleoSpin miRNA kit.
This will surely help you to get better yield.
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