A: The Total Exosome RNA and Protein Isolation Kit is developed specifically for exosome samples, and uses an initial Acid-Phenol: Chloroform extraction. This is followed by addition of ethanol to the aqueous phase, and then the sample is passed through a glass-fiber containing filter cartridge to immobilize the RNA. The filter is washed, and the RNA is eluted with a low ionic-strength solution. The kit recovers all RNA longer than ~10 nt up to several kb. (The majority of exosomal RNA ranges in size from ~20–300 nt.) The product manual also describes an additional protocol for enrichment of short RNA (<200 nt), but we recommend the total RNA isolation protocol in order to maximize recovery of all RNA, including various mRNA, rRNA, and ncRNA fragments.
The kit also provides an option to recover protein from the same sample through the use of the Exosome Resuspension Solution. The kit can be used to isolate RNA and protein from exosomes purified from any sample type using either the Total Exosome Isolation reagents or any other protocol such as ultracentrifugation.
A: No, the described effect does not have a negative effect on the RNA recovery.
A: This can vary depending on the sample type, volume of sample, isolation method, and exosome content/concentration. Listed below are some examples:
- When exosomes are isolated from 30 ml of HeLa cell culture media using the Total Exosome Isolation (from cell culture media) reagent, it is possible to recover ~8 ng exosomal RNA.
- For exosomes recovered from 4 ml serum using the Total Exosome Isolation (from serum) reagent, ~2 ng exosomal RNA can be obtained.
In both cases, these amounts of RNA are sufficient for RNA library prep for Ion Torrent PGM or Proton sequencing. For real-time PCR analysis, substantially smaller amounts of RNA are needed and much lower sample volumes can be utilized. For example, RNA recovered from 3 μl serum or 30 μl cell media is enough for one RT-qPCR reaction.