TY - JOUR T1 - Single-step RT-qPCR for detection of extracellular vesicle microRNAs in vivo: a time- and cost-effective method. AU - Lee,Heedoo, AU - He,Xue, AU - Le,Trung, AU - Carnino,Jonathan M, AU - Jin,Yang, Y1 - 2020/02/19/ PY - 2020/2/20/pubmed PY - 2020/7/17/medline PY - 2020/2/20/entrez KW - extracellular vesicle KW - microRNA KW - single-step RT-qPCR SP - L742 EP - L749 JF - American journal of physiology. Lung cellular and molecular physiology JO - Am J Physiol Lung Cell Mol Physiol VL - 318 IS - 4 N2 - Emerging evidence suggests that extracellular vesicle (EV)-associated microRNAs (miRNAs) are a potential diagnostic tool for liquid biopsy in various human diseases. However, the experimental procedure for the detection of EV-associated miRNAs (EV-miRNAs) from body fluids is relatively complex and not cost-effective. Due to the limited amount of EVs and EV-RNAs, a column-based RNA purification, which is an expensive approach, is often used to detect EV-miRNAs via reverse transcription-quantitative real-time PCR (RT-qPCR). Here, we developed and validated a simple and cost-effective method (single-step RT-qPCR) in which we directly detect EV-miRNAs without RNA purification from the EVs. We validated this protocol using the EVs isolated from mouse broncho-alveolar lavage fluid (BALF) and serum. The obtained EVs were first lysed in the EV-lysis buffer, followed by RT-qPCR without isolation and purification of RNAs. We successfully detected the designated miRNAs from lysed EVs; 106 to 107 EVs were optimal to detect the EV-miRNAs using the single-step RT-qPCR. In our previously published work, using the conventional RT-qPCR method, we have reported that miR-142 and -223 are dramatically upregulated in both BALF and serum EVs after lung infection. Hence, we reassessed and confirmed the level of EV-miR-142/223 using the newly developed single-step RT-qPCR. Notably, inhibition of RNase activity in the lysed EVs remains crucial for the detection of EV-miRNAs. Moreover, repeated freeze-thaw cycling significantly interferes the EV-miRNA quantification. Collectively, the single-step RT-qPCR for the detection of EV-miRNAs in vivo will potentially provide a fast, accurate, and convenient way to quantify circulating and/or body fluid-derived EV-miRNAs. This method may potentially be applied to the diagnostic blood testing used in the medical centers or research laboratories. SN - 1522-1504 UR - https://www.unboundmedicine.com/medline/citation/32073880/Single_step_RT_qPCR_for_detection_of_extracellular_vesicle_microRNAs_in_vivo:_a_time__and_cost_effective_method_ DB - PRIME DP - Unbound Medicine ER -