Data & Figures

EXO-NET Data & Figures

  EXO-NET isolated plasma EV characterisation

Objective: To confirm the presence of known EV protein and RNA markers in EXO-NET isolated EVs from human plasma.

MethodPooled normal human plasma (500µl) was incubated with 30 µl of EXO-NET for 15 min. EXO-NET-captured EVs were magnetically isolated and lysed for downstream analysis. Total EV protein and RNA were extracted and analysed by Western blot and qPCR.

Result: Western blot analysis showed EXO-NET-isolated EVs from normal human plasma were positive for CD63, CD81, CD9, TSG101 and Flotillin-1, and negative for calnexin (an EV negative marker). qPCR analysis demonstrated that EXO-NET isolated EVs had high yield of mRNA (GAPDH) and microRNA (miR-191).

  EXO-NET isolated saliva EV characterisation

ObjectiveTo confirm the presence of known EV protein and RNA markers in EXO-NET isolated EVs from human saliva.

MethodPooled normal human saliva (400µl) was diluted with 400µl of PBS and then incubated with 30 µl  of EXO-NET for 15 min.  EXO-NET captured EVs were magnetically isolated and lysed for downstream analysis.  Total protein and RNA were extracted from captured EVs and analysed by Western blot and qPCR.

Result: Western blot analysis confirmed the presences of CD63, CD81 and CD9. Calnexin (EVs negative marker) was not detectable. qPCR analysis confirmed that EXO-NET isolated EVs had high yield of mRNA (GAPDH) and microRNA (U6).

  EXO-NET isolated cell conditioned medium EV characterisation

 

Objective: To confirm the presence of known EV protein and RNA markers in EXO-NET isolated EVs from MCF-7 cell conditioned medium.

Method: MCF-7 cells were cultured in growth medium (MEM) + 10% FBS + 1X GlutaMAXTM Supplement) to ~80% confluency. Cells were incubated in serum free Essential 8 (E8) media or basal MEM media supplemented with 1X GlutaMAXTM (MEG) for 24 h.  Cell-conditioned medium (CCM, 20mL) was incubated with 30 µl of EXO-NET for 30 min.  EXO-NET captured EVs were magnetically separated from the CCM.

Result: Western blot analysis confirmed presence of CD9 and Flotillin-1.  qPCR demonstrated that EXO-NET isolated EVs had higher yield of mRNA (GAPDH) and microRNA (U6 and miR191) in E8-CCM than MEG-CCM.

  EV isolation comparison | RNA

Objective: To compare mRNA and microRNA yield and recovery from EXO-NET isolated EVs with 4 other EV isolation kits.

Method: EVs were isolated from pooled normal human plasma by EXO-NET and other 4 commercial kits according to their manufacturers’ instructions.  Isolated EVs were lysed and total RNA were extracted for qPCR analysis to measure both microRNAs (miR-16, let-7a and miR-21) and mRNAs (GAPDH, OAZ1, RPLPO and SERF2).

Result: EXO-NET delivers equivalent or higher recovery of plasma EV RNA (mRNAs and microRNAs) compared to other 4 commercial EV isolation kits as indicated by a lower CT value.  *RNA level was below the limit of detection.

  EV isolation comparison | Protein

Objective: To compare mass spectrometry proteomic analysis of EXO-NET isolated EVs with another bead-based EV isolation kit.

Method: EVs were isolated from pooled normal human plasma by EXO-NET and Kit A according to manufacturers’ instructions.  Isolated EVs were lysed and extracted total protein were processed for proteomic analysis by tandem mass spectrometry.

Result: Mass spectrometry analysis showed that albumin peptide intensity in Kit A was 2-fold higher than that observed for EXO-NET isolated EVs.  EXO-NET reduces contamination of EV preparations by serum proteins that may confound downstream analysis. The EV peptides intensity in Kit A was 6 to 10-fold less than that observed for EXO-NET-isolated EVs.