Exosome Analysis

Standard housekeeping genes aren’t reliable in exosomes. Use synthetic spike-in controls added at extraction (e.g., C. elegans miRNA) or stable small RNAs (like miR-16) for consistent normalization.

A clear, sticky pellet often means low input or pellet loss. Ensure ≥2 mL plasma, handle pellets gently (avoid over-washing), and check for RNase contamination. If Bioanalyzer shows nothing, verify pellet resuspension and buffer compatibility.

Cytosolic proteins like actin/GAPDH are often absent. Rely instead on exosome-enriched markers such as CD63, CD9, CD81 or TSG101. If TSG101 is weak, CD63 is a robust alternative.

Gently resuspend in warm (37 °C) buffer, pipetting slowly. For stubborn pellets, add a small amount of PBS with 0.01 % Tween-20 to ease dispersion—just avoid detergents that interfere with MS.

Repeated freeze–thaw can disrupt membrane integrity and alter lipid composition. Store aliquots at –80 °C and minimize thaw cycles; consider cryoprotectants like trehalose for better lipid preservation.

Filter samples (0.22 µm), dilute appropriately, and avoid excessive freezing/thawing. Adding a carrier protein (e.g., 0.1 mg/mL BSA) helps stabilize vesicles and reduce aggregates.

Yes, serum-derived vesicles or particulate contaminants in the media can be present. Always run a media‐only blank to identify and subtract background counts.

Pre-quantify exosomes (BCA/NTA) to standardize input. Use bead-based capture (anti-CD9/63 beads), stain with fluorescent antibodies, set voltages to detect small events, and include unstained/isotype controls for accurate gating.