Prostate cancer antigen 3(PCA3) is a long non-coding RNA, which was found increased expression in CaP patients than healthy individual. In this study, the individual nucleic acid of PCA3 and PSA was recombinant expressed as a reference reagent, and a quantitative RT-PCR with TaqMan assay was developed to examine the copies of PCA3 and PSA gene in urine. The results showed that the area under the receiver operating characteristic curve (AUROC) was 0.717, 0.444 and 0.916 for the number of PCA3 copy, PSA copy and for the score of PCA3/PSA RNA, respectively. Additionally, the AUROC for serum tPSA was 0.674 with a low specificity of 12.07%. Finally, the algorithm of PCA3 RNA versus PSA RNA was evaluated and corroborated as CaP biomarker by conducting a multicentric clinical trial. This study not only validated the developed technique of qRT-PCR with TaqMan assay for examination of urinary PCA3 and PSA RNA, it also demonstrated that the score of the PCA/PSA RNA was a reliable signature for CaP diagnosis.

Aim To assess the exosomal miR‐21/Let‐7a ratio, a noninvasive method, in distinguishing non‐small cell lung cancer (NSCLC) from benign pulmonary diseases. Methods The exosomes were extracted from the peripheral blood serum using serum exosomal extraction kit. miR‐21 and Let‐7a levels were evaluated by quantitative reverse transcription polymerase chain reaction. Results We found that miR‐21/Let‐7a ratio of NSCLC patients was significantly higher than that of healthy people, patients with pulmonary inflammation diseases, and benign pulmonary nodules, respectively. Receiver‐operating characteristic analysis revealed that as compared with healthy controls, miR‐21/Let‐7a produced the area under the curve (AUC) at 0.8029 in patients with NSCLC, which helped to distinguish NSCLC from healthy controls with 81.33% sensitivity and 69.57% specificity. In addition, the AUC of miR‐21/Let‐7a in NSCLC patients was 0.8196 in comparison to patients with pulmonary inflammation diseases. Meanwhile, the sensitivity and specificity were 56.00% and 100%, respectively. Furthermore, compared with patients with benign pulmonary nodules, the AUC of miR‐21/Let‐7a in NSCLC patients was 0.7539. The sensitivity and specificity were 56.00% and 82.61%, respectively. Conclusion In the present study, our findings revealed that exosomal miR‐21/Let‐7a ratio holds considerable promise as a noninvasive biomarker for the diagnosis of NSCLC from benign pulmonary diseases.

Background Exosomes are nano-sized extracellular vesicles containing different biomolecules such as proteins and microRNAs (miRNAs) that mediate intercellular communication. Recently, numerous studies have reported the important functions of exosomal miRNAs in disease development and the potential clinical application as diagnostic biomarkers. Up to now, the most commonly used methods to extract exosomes are ultracentrifugation (UC) and precipitation-based commercial kit (e.g., ExoQuick). Generally, both UC and ExoQuick method could co-isolate contaminating proteins along with exosomes, with the UC method yielding even purer exosomes than ExoQuick. However, the comparison of these two methods on co-precipitated free miRNAs is still unknown. Methods In this study, we isolated exosomes from the human serum with exogenously added cel-miR-39 by UC and ExoQuick and compared the proportion of cel-miR-39 co-precipitated with exosomes extracted by these two methods. Results Using exogenous cel-miR-39 as free miRNAs in serum, we concluded that ExoQuick co-isolates a small proportion of free miRNAs while UC hardly precipitates any free miRNAs. We also found that incubation at 37 °C for 1 h could decrease the proportion of free miRNAs, and exosomal miRNAs like miR-126 and miR-152 also decreased when RNase A was used. In conclusion, our findings provide essential information about the details of serum exosome isolation methods for further research on exosomal miRNAs.

BackgroundExosomes are nano-sized extracellular vesicles containing different biomolecules such as proteins and microRNAs (miRNAs) that mediate intercellular communication. Recently, numerous studies have reported the important functions of exosomal miRNAs in disease development and the potential clinical application as diagnostic biomarkers. Up to now, the most commonly used methods to extract exosomes are ultracentrifugation (UC) and precipitation-based commercial kit (e.g., ExoQuick). Generally, both UC and ExoQuick method could co-isolate contaminating proteins along with exosomes, with the UC method yielding even purer exosomes than ExoQuick. However, the comparison of these two methods on co-precipitated free miRNAs is still unknown.ResultsIn this study, we isolated exosomes from the human serum with exogenously added cel-miR-39 by UC and ExoQuick and compared the proportion of cel-miR-39 co-precipitated with exosomes extracted by these two methods. Using exogenous cel-miR-39 as free miRNAs in serum, we concluded that ExoQuick co-isolates a small proportion of free miRNAs while UC hardly precipitates any free miRNAs. We also found that incubation at 37℃ for 1 h could decrease the proportion of free miRNAs, and exosomal miRNAs like miR-126 and miR-152 also decreased when RNase A was used.ConclusionsIn conclusion, our findings provide essential information about the details of serum exosome isolation methods for further research on exosomal miRNAs.