This study investigates the distinct RNA profiles of three subpopulations of extracellular vesicles (EVs): apoptotic bodies (ABs), microvesicles (MVs), and exosomes (EXOs). Using two centrifugation-based protocols, EVs were isolated from three cell lines (HMC-1, TF-1, and BV-2). RNA profiles were analyzed using a Bioanalyzer, revealing that ribosomal RNA (rRNA) was primarily detectable in ABs, while EXOs contained smaller RNAs without prominent rRNA peaks. MVs from HMC-1 and BV-2 cells contained little or no RNA, except for those from TF-1 cells. Transmission electron microscopy (TEM) confirmed the distinct morphological characteristics of ABs, MVs, and EXOs. Flow cytometry showed that CD63 and CD81 were present in all EVs, except CD9 in TF-1-derived vesicles. The study concludes that centrifugation-based protocols are effective for distinguishing EV subpopulations, as they show different RNA profiles and morphological features. However, CD63-coated beads cannot distinguish between ABs, MVs, and EXOs in flow cytometry analysis. The results highlight the importance of proper isolation methods to avoid contamination and accurately characterize EVs.This study investigates the distinct RNA profiles of three subpopulations of extracellular vesicles (EVs): apoptotic bodies (ABs), microvesicles (MVs), and exosomes (EXOs). Using two centrifugation-based protocols, EVs were isolated from three cell lines (HMC-1, TF-1, and BV-2). RNA profiles were analyzed using a Bioanalyzer, revealing that ribosomal RNA (rRNA) was primarily detectable in ABs, while EXOs contained smaller RNAs without prominent rRNA peaks. MVs from HMC-1 and BV-2 cells contained little or no RNA, except for those from TF-1 cells. Transmission electron microscopy (TEM) confirmed the distinct morphological characteristics of ABs, MVs, and EXOs. Flow cytometry showed that CD63 and CD81 were present in all EVs, except CD9 in TF-1-derived vesicles. The study concludes that centrifugation-based protocols are effective for distinguishing EV subpopulations, as they show different RNA profiles and morphological features. However, CD63-coated beads cannot distinguish between ABs, MVs, and EXOs in flow cytometry analysis. The results highlight the importance of proper isolation methods to avoid contamination and accurately characterize EVs.