Open AccessSingle-step RT-qPCR for detection of extracellular vesicle microRNAs in vivo: a time- and cost-effective method
Author(s) -
Heedoo Lee,
Xue He,
Trung Le,
Jonathan M. Carnino,
Yang Jin
Publication year - 2020
Publication title -
american journal of physiology. lung cellular and molecular physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.892
H-Index - 163
eISSN - 1522-1504
pISSN - 1040-0605
DOI - 10.1152/ajplung.00430.2019
Subject(s) - microrna , extracellular vesicle , lysis , lysis buffer , real time polymerase chain reaction , rna , rna extraction , biology , microbiology and biotechnology , reverse transcriptase , rnase p , extracellular vesicles , microvesicles , liquid biopsy , computational biology , chemistry , gene , biochemistry , genetics , cancer
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; 10 6 to 10 7 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.