Impact of Extracellular Vesicle Isolation Methods on Downstream Mirna Analysis in Semen: A Comparative Study

Abstract

Seminal plasma (SP) contains a unique concentration of miRNA, mostly contained in small extracellular vesicles (sEVs) such as exosomes, some of which could be clinically useful for diagnosis and/or prognosis of urogenital diseases such as prostate cancer (PCa). We optimized several exosome-EV isolation technologies for their use in semen, evaluating EV purifying effectiveness and impact on the downstream analysis of miRNAs against results from the standard ultracentrifugation (UC) method to implement the use of SP sEV_miRNAs as noninvasive biomarkers for PCa. Our results evidenced that commercial kits designed to isolate exosomes/EVs from blood or urine are mostly applicable to SP, but showed quantitative and qualitative variability between them. ExoGAG 3500× g and the miRCURY Cell/Urine/CSF 1500× g methods resulted as equivalent alternative procedures to UC for isolating exosomes/sEVs from semen for nanoparticle characteristics and quality of RNA contained in vesicles. Additionally, the expression profile of the altered semen sEV-miRNAs in PCa varies depending on the EV isolation method applied. This is possibly due to different extraction techniques yielding different proportions of sEV subtypes. This is evidence that the exosome-EV isolation method has a significant impact on the analysis of the miRNAs contained within, with important consequences for their use as clinical biomarkers. Therefore, miRNA analysis results for EVs cannot be directly extrapolated between different EV isolation methods until clear markers for delineation between microvesicles and exosomes are established. However, EV extraction methodology affects combined models (semen exosome miRNA signatures plus blood Prostate specific antigen (PSA) concentration for PCa diagnosis) less; specifically our previously described (miR-142-3p + miR-142-5p + miR-223-3p + PSA) model functions as molecular marker from EVs from any of the three isolation methods, potentially improving the efficiency of PSA PCa diagnosis.

Keywords: biomarker; diagnosis; exosomes; extracellular vesicle isolation methods; extracellular vesicle miRNA analysis; extracellular vesicles; microvesicles; prostate cancer; semen.

Figure 1

Technical conditions of the different exosome isolation methods applied. Semen-derived small extracellular vesicles (sEVs), including exosomes, were isolated using four precipitation-based reagents to compare with ultracentrifugation as a gold-standard technique. Different incubation and centrifugation conditions were tested. miRCURY S/P: miRCURY Exosome Serum/Plasma kit; miRCURY _Cell_U_CSF: miRCURY Exosome Cell/Urine/CSF kit; ExoQuick Ultra S/P: ExoQuick Ultra EV Isolation kit for Serum and Plasma; ExoGag: ExoGAG kit; UC: ultracentrifugation protocol; Pp: precipitation step.

Figure 2

Nanoparticle tracking analysis (NTA) results of the EVs extracted from human semen using the different isolation methods and conditions. (A) Particle size is shown as the mode of EV diameter and (B) EV concentration is shown as the log10 of the number of particles/mL of the extracts obtained. Error bars represent the standard deviation (SD) of the mode/mean. (C) Representative NTA images of isolated vesicles. ExoQuick Ultra A refers to the protocol which includes precipitation and the purification columns whereas ExoQuick Ultra B only includes precipitation.

Figure 3

Characterization of sEVs by tetraspanin composition. sEVs isolated with ExoGAG (ExoGAG 3500× g), ultracentrifugation and miRCURY (miRCURY Exosome Cell/Urine/CSF 1500× g) protocols were tested. (A) Representative profiles of Alexa 488 fluorescence obtained by cytometry analysis. IgG (immunoglobulin G) was used as negative control of fluorescence. Positive CD63 and CD81 labelling was represented by a displacement of fluorescence peak curve. (B) Quantification of fluorescence represented by a fold-change of fluorescence intensity median with respect to IgG negative control. A specific CD63 and CD81 labelling of SP EVs isolated by ExoGAG is observed. A similar CD63 labelling was found when EVs were purified by ultracentrifugation, although a higher CD81 signal was obtained when compared with ExoGAG EVs. EVs isolated with miRCURY EV/exosome isolation kit showed a CD81 positive labelling.

Figure 4

Semen exosome miR-30d-5p, miR-93-5p and miR-449a Cp values obtained by RT-qPCR. RNA was obtained from extracellular vesicle extracts isolated by using different techniques: ExoGAG (3500× g), miRCURY Cell/Urine/CSF (1500× g) and Ultracentrifugation (100,000× g) methods. First-stranded cDNA specific for miRNA was obtained by RT of 50 µg in 10 µL using the Universal cDNA synthesis kit (Exiqon). The three semen exosomal miRNAs were amplified in a Lightcycler 96 instrument. No difference in the Cp values were found between the three methods. The values in the graph are the mean Cp values ± SD. RT-qPCR: quantitative reverse transcription PCR.

Figure 5

Expression profiling of the sixteen miRNAs (A, let7i-3p, B, miR-106b-5p, C, miR-125a-5p, D, miR-130a-3p, E, miR-142-3p, F, miR-142-5p, G, miR-181c-5p, H, miR-196b-3p, I, miR-223-3p, J, miR-30c-5p, K, miR-30e-3p, L, miR-34a-3p, M, miR-34a-5p, N, miR-576-5p, O, miR-663b, P, miR-92a-3p) in semen EV isolated by three methods: UC (ultracentrifugation), ExoGAG and miRCURY (miRCURY Exosome Cell/Urine/CSF 1500× g). MiRNA expression levels were determined by RT-qPCR in healthy controls (HCt), benign prostate hyperplasia (BPH), and prostate cancer affected men (PCa). Data are shown as RQ values, which were calculated using the 2^dCp strategy and relative to the mean expression values of miR-576-5p and miR-181c-5p. The horizontal bar displays the median cellular expression level. RT-qPCR: quantitative reverse transcription PCR.

Figure 5

Expression profiling of the sixteen miRNAs (A, let7i-3p, B, miR-106b-5p, C, miR-125a-5p, D, miR-130a-3p, E, miR-142-3p, F, miR-142-5p, G, miR-181c-5p, H, miR-196b-3p, I, miR-223-3p, J, miR-30c-5p, K, miR-30e-3p, L, miR-34a-3p, M, miR-34a-5p, N, miR-576-5p, O, miR-663b, P, miR-92a-3p) in semen EV isolated by three methods: UC (ultracentrifugation), ExoGAG and miRCURY (miRCURY Exosome Cell/Urine/CSF 1500× g). MiRNA expression levels were determined by RT-qPCR in healthy controls (HCt), benign prostate hyperplasia (BPH), and prostate cancer affected men (PCa). Data are shown as RQ values, which were calculated using the 2^dCp strategy and relative to the mean expression values of miR-576-5p and miR-181c-5p. The horizontal bar displays the median cellular expression level. RT-qPCR: quantitative reverse transcription PCR.