# Isolation of Exosomes from MDA-MB-231 Cells Using a Paddle Screw System and Detection of TNBC-Associated Exosomal miRNAs

## TL;DR

This study presents a new method to isolate exosomes from breast cancer cells and detect cancer-related miRNAs using a mechanical system and sensitive assays.

## Contribution

A novel paddle screw system for exosome isolation and a bidirectional extension assay for miRNA detection are integrated for scalable cancer diagnostics.

## Key findings

- Exosomes were rapidly and efficiently isolated from MDA-MB-231 cells using a paddle screw system.
- The BDE assay improved PCR specificity and reduced background signals for miRNA detection.
- The method showed high sensitivity, wide dynamic range, and reproducibility for TNBC-associated miRNAs.

## Abstract

Exosomes are nanoscale extracellular vesicles that carry disease-associated microRNAs (miRNAs) and represent promising biomarkers for cancer diagnosis. Triple-negative breast cancer (TNBC) lacks well-defined molecular markers, necessitating sensitive and integrable analytical approaches for TNBC-related exosomal miRNAs. In this study, exosomes were isolated from MDA-MB-231 TNBC cells using a paddle screw-based system designed to enhance mass transfer through active rotation, providing a mechanically driven isolation strategy that is compatible with miniaturized and microfluidic platforms. This dynamic isolation process enabled rapid and efficient exosome recovery within a short processing time. Three TNBC-associated miRNAs encapsulated in the isolated exosomes were quantitatively analyzed using polyadenylation tailing (poly(A) tailing) and specific bidirectional extension sequence-based assays combined with reverse transcription quantitative real-time PCR (RT-qPCR). The bidirectional extension (BDE) assay generated highly specific PCR templates, leading to improved amplification specificity and reduced background signals. The RT-qPCR analysis exhibited high sensitivity, wide dynamic range, and good reproducibility for all target miRNAs. Overall, these results demonstrate that the integration of a paddle screw-based exosome isolation module with an extension-based nucleic acid detection strategy provides a scalable and biosensor-compatible analytical framework for profiling TNBC-associated exosomal miRNAs, with potential applications in microfluidic liquid biopsy platforms and exosome-based cancer diagnostics.