Collection of human blood and plasma preparation

EDTA-Blood was collected from healthy donors and processed for plasma isolation immediately after collection. Blood plasma was centrifuged at 14000g for 10min and diluted 1/10 in 1×PBS (w/o Ca, Mg) before filtration through 0.22μm filters (Millex®GS, Millipore), ulftrafiltration, ultracentrifugation and RNA isolation. This study has been approved by the ethics committee, Heidelberg, Germany.

Preparation of conditioned media, ultracentrifugation and ultrafiltration

To obtain conditioned media for RNA isolation, ultracentrifugation and ultrafiltration, MCF7 cells were seeded in culture flasks and the media was renewed ~12h after plating. After 3 days in culture, media were collected and centrifuged at 1200g for 3min at RT to remove living cells and subsequently centrifuged at 14000g for 10min at RT to remove cell debris. For the ultracentrifugation, 6ml of conditioned media were combined with 6ml of DMEM and the resulting 12ml were loaded into the ultracentrifugation tube. Ultracentrifugation of blood plasma (diluted 1/10 in PBS) and conditioned media was performed at 110000g for 2h at 4°C. The 110000 g pellets were washed twice with 1×PBS and then dissolved in 700μl of Qiazol reagent for total RNA isolation (Qiagen). Ultrafiltration of blood plasma (diluted 1/10 in PBS) and conditioned media was performed using Vivaspin20 columns (Sartorius Stedim GmbH) with 300, 100 and 50kDa MWCO according to the manufacturer’s instructions.

Isolation of miRNAs

Isolation of extracellular miRNAs from liquid samples was performed using Tri-Reagent LS (Sigma) together with miRNeasy kit (Qiagen). Briefly, 1.2ml of Tri-Reagent LS were added to 0.4ml of blood plasma or conditioned media, mixed by vigorous shaking for 10s and then incubated for 10min at RT to ensure complete dissociation of nucleoprotein complexes. Then (i) 5pg of synthetic miRNA-39 from Caenorhabditis elegans (cel-miRNA-39) were added as a spike-in control for purification efficiency; (ii) 1.2μl of glycogen (10mg/ml) were added to enhance the efficiency of RNA column binding. After supplying 320μl of chloroform, the mixture was vigorously shaken for 45s and allowed to stand for 5min at RT. Following the centrifugation at 14 000g for 20min total RNA was precipitated from the upper (aqueous) phase by adding 1.5 V of 100% ethanol. Purification of extracted total RNA was performed with miRNeasy columns (Qiagen) according to the manufacturer’s instructions. From the Qiagen columns, RNA was eluted in 30μl of RNase-free water. Total RNA from cells monolayers grown on 24-well plates was isolated using Qiazol reagent (Qiagen) in combination with miRNAeasy kit (Qiagen) according to the manufacturer’s protocol. To monitor the extraction efficacy, 5pg of cel-miR-39 was added to the cell/Qiazol lysates. RNA was eluted in 30μl of RNase-free water.

Quantitative real-time PCR

A fixed volume of 5μl of total RNA from 30μl eluates obtained after RNA isolation of a given sample was used as an input into a reverse transcription (RT) reaction. The levels of α-tubulin and β-actin mRNAs were measured using corresponding TaqMan Gene Expression Assays (Applied Biosystems) according to the manufacturer’s instructions. The levels of mature hsa-miR-16, hsa-miR-21, hsa-miR-24, hsa-miR-34b, cel-miR-39 mRNAs were measured using individual TaqMan microRNA Assays (Applied Biosystems) according to the manufacturer’s instructions with minor modifications. Cel-miR-39 was used for the normalization of both extracellular and intracellular miRNAs levels. Since we compared intracellular and extracellular expression of miRNAs in the same cell line it was possible to use only cel-mir-39 as spiked-in control for the normalization of miRNAs in the cells as well as in the media. Reverse transcriptase reactions contained 5μl of purified total RNA, 50/NnM stem–loop RT primers (where N is a number of analysed miRNAs), 1×RT buffer, 0.25mM each of dNTPs, 3.33U/µl MultiScribe reverse transcriptase and 0.25U/µl RNase inhibitor. RT reaction had final volume of 15µl were incubated for 30min at 16°C, 30min at 42°C, 5min at 85°C and then held at 4°C. RT product was further diluted six times with RNAse-free water. Real-time PCR was performed using Taqman Universal PCR Master Mix. A 10µl PCR reaction included 2µl of diluted RT product, 1×TaqMan Universal PCR Master Mix (Applied Biosystems) and 1×of the corresponding miRNA assay primers. The reactions were incubated in 384-well plates at 95°C for 10min, followed by 50 cycles of 95°C for 15s and 60°C for 1min. All reactions were run in duplicate. Real-time PCR was performed using the LightCycler 480 Real-Time PCR System (Roche). Data was analyzed with the LightCycler 480 software (Roche), determining the threshold cycle (C_t) by the second derivative max method. Relative quantities of miRNA were calculated using the ΔΔC_t method after normalization to the cel-miR-39 spiked-in control.

miRNA profiling

The expression of miRNA in the blood plasma, MCF7 cells, MCF7 cells conditioned media, control DMEM+ 10% FBS and 110000 g pellets was profiled using stem–loop RT–PCR-based TaqMan Human MicroRNA Arrays (Applied Biosystems). The arrays represented 667 mature miRNAs and miRNAs* present in the Sanger miRBase v12 in a two-card set of arrays (Array A v2.1 and Array B v2.0). RT-PCR reactions were performed according to the manufacturer’s instructions. Briefly, total RNA (3μl per reaction) was reverse transcribed using TaqMan miRNA Reverse Transcription Kit (Applied Biosystems) in combination with the stem–loop Megaplex primers pool sets A and B in total volume of 7.5μl. The RT cycling conditions were: 40 cycles of 16°C for 2min, 42°C for 1min and 50°C for 1s. Megaplex RT products were further pre-amplified using TaqMan PreAmp Master Mix and Megaplex PreAmp primers (Applied Biosystems). Briefly, 2.5μl of the megaplex RT products were mixed with 2.5μl of Megaplex PreAmp Primers (pool A or B) and 12.5μl TaqMan PreAmp Master Mix in a 25μl PCR reaction. The pre-amplification cycling conditions were 95°C for 10min, 55°C for 2min and 75°C for 2min followed by 12 cycles of 95°C for 15s and 60°C for 4min. The pre-amplified cDNA was diluted with 0.1× TE (pH 8.0) to 100μl and 10μl of diluted cDNA was used in each plate for real-time PCR reactions. The expression profiles of miRNA were acquired using TaqMan Low-Density Arrays. Briefly, quantitative real-time PCR was carried out on Applied BioSystems 7900HT thermocycler using the manufacturer’s recommended cycling conditions. Cycle threshold (C_t) values were calculated with the SDS software v2.3 using automatic baseline settings with assigned minimum threshold of 0.2. Because of the fact that the C_t value of 35 represents single template detection, C_t value >35 were considered to be below the detection level of the assays (17). Therefore, all miRNAs with C_t>35 were included in the analysis as ‘undetected’ and assigned the C_t values of 40. Array experiments were performed only once using samples from one biological experiment. Those miRNAs that appeared on the DMEM+10% FBS (negative control sample) array were removed from the analysis (for all miRNAs that were more than 1.5 Δ C_t, as compared to conditioned media samples).

Immunoprecipitation

Immunoprecipitation of extracellular miRNAs with anti-Ago2 antibody (Abnova, clone 2E12-1C9; cat. H00027161-M01) was performed using Pierce Classic IP kit (Thermo Scientific). Binding with anti-Ago2 was carried out in a detergent-free solution. The maximal efficiency of immunoprecipitation was achieved when 5μg of anti-Ago2 antibody was combined with 5μl of sample. Briefly, 10μl of 1×PBS were added to 5μl of 2× concentrated human blood plasma (or 10× concentrated conditioned media) to form 15μl of miRNA binding solution. The concentration of the samples was performed using Vivaspin2 columns with 10kDa MWCO (Sartorius Stedim GmbH). To form Ago2/anti-Ago2 complexes, 15μl of corresponding miRNA binding solutions were combined with 5μg (10μl) of anti-Ago2 antibody and incubated overnight at +4°C. To capture the immune complexes, 20μl Pierce Protein A/G Agarose suspended in 200μl of 1×PBS was added to the antibody/lysate samples and incubated for 2h on a shaker at RT. The A/G Agarose resin was washed two times with 1ml of IP Lysis/Wash buffer. Afterwards, 700μl of Qiazol reagent was added to the resin and total RNA was isolated with the miRNeasy kit (Qiagen) as described above. Cel-miR-39 (5pg) was spiked into the samples after addition of Qiazol reagent and used as normalization control. Immunoprecipitation of exogenously expressed FLAG-fused Ago proteins was approached similarly, except that 100μl of 10× concentrated conditioned media was combined with 200μl of Pierce IP Lysis buffer (0.025 M Tris, 0.15 M NaCl, 0.001 EDTA, 1% NP-40, 5% glycerol, pH 7.4) before incubating with 10μg of mouse anti-FLAG antibody (Sigma, clone M2).

Most of the extracellular miRNAs are not associated with exosomes

Extracellular miRNAs have been previously detected in cell-exported exosomes, small membrane vesicles of 50–100nm in diameter (12,14–16). However, to our knowledge, no experiments demonstrating that extracellular miRNA is associated exclusively with exosomes have been described. We have noticed that incubation of either blood plasma or conditioned media with the 1% NP-40 detergent did not restore the nuclease sensitivity of extracellular miRNAs (data not shown). To further confirm the hypothesis of exosome-free extracellular miRNA, we fractionated blood plasma and conditioned media of MCF7 cells according to well-developed methods for exosomes isolation (12,18). Thus, cell debris and microvesicles were removed from the samples by filtration through 0.22μm filters; the exosome-free fractions were obtained by further ultracentrifuging the samples at 110000g for 2h at +4°C (Figure 2A and B). The levels of three miRNA species (hsa-miR-16, hsa-miR-21 and hsa-miR-24) were quantitatively assayed in the samples after each fractionation step with individual TaqMan miRNA Assays. To our surprise, ultracentrifugation did not significantly decrease the level of any of these miRNAs in both blood plasma and conditioned media (Figure 2C and D). Accordingly, 110000g pellets contained only trace amounts of the respective miRNAs (blood plasma: miR-16: 0.34±0.07%; miRNA-21: 0.21±0.14%; miRNA-24: 0.94±0.68%; MCF7 media: miR-16: 1.57±0.5%; miRNA-21: 1.9±1.18%; miRNA-24: 1.19±0.80%). The presence of exosomal marker protein CD9 in 110 000g pellets was confirmed by western immunoblot analysis (Figure 2B). Furthermore, TaqMan Low Density Array (Applied Biosystems) of 707 different miRNAs and miRNAs* performed on total RNAs isolated from 400μl (1/30V) of 110000g supernatants and total 110000g pellets revealed that only a small proportion of all miRNAs was specifically associated with the pellet fractions in both plasma and conditioned media (Figure 3). Those miRNAs that appeared on the profile with C_t value of <35 were considered as ‘detected’. In the conditioned media the average ΔC_t of miRNAs detected in 1/30V supernatant and 1V pellet was −1.6 indicating ~90 times higher miRNA amounts in the supernatant compared to pellet (Figure 3A). Extracellular miRNAs that were found exclusively in the exosomes-free supernatant and in the exosomes-containing pellet are listed in Supplementary Tables S2 and S3. Out of 188 miRNA species detected in the blood plasma, 97 miRNAs were detected both in the pellet and supernatant (Figure 3B) and 69 miRNA species were found exclusively in the supernatant (Figure 3B, ES; Supplementary Table S4). Only 22 different miRNAs were detected exclusively in the pellet fraction of blood plasma (Figure 3B, EP; Supplementary Table S5), however, the C_t values of these miRNAs ranged from 30.8 to 34.9. The average ΔC_t for miRNAs between 1/30 V supernatant and 1 V pellet was −1.4 indicating that the amount of miRNA was ~80 times higher in the plasma supernatant compared to the pellet. Therefore, our data strongly indicates that the level of miRNAs that can be associated with exosomes is dramatically lower compared to the amount of exosomes-free miRNAs per volume of blood plasma or conditioned media.

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Figure 2.

(A) The procedure for the fractionation of human blood plasma and conditioned media. Cell-debris free (1200g following 14000g centrifugation) blood plasma diluted 12 times in PBS and MCF7 cells conditioned media were used as starting samples. Microvesicles were removed from the samples by filtration through 0.22μm filters; exosome-free fractions were obtained by ultracentrifugation at 110000g. Furthermore, fractionation of 0.22μm filtered samples was performed using 300, 100 and 50kDa MWCO nanomembranes. (B) The efficacy of ultracentrifugation to remove exosomes and large proteins from plasma was assessed by SDS–PAGE gel of the 110000g pellets. Coomassie blue (left) staining and western immunoblot (right) had shown that the first ultracentrifugation (PI pellet) removed large proteins (>300kDa) and exosomes (as judged by exosomal marker protein CD9) from the plasma supernatant. While the pellet obtained after second ultracentrifugation of the same supernatant (PII) at the same force did not contain any detectable amounts of proteins including exosomal marker. (C and D) The level of three miRNA species miR-16, miR-21 and miR-24 was quantitatively assayed in the conditioned media (C) and blood plasma (D) samples after each fractionation step with individual TaqMan miRNA Assays. Spiked in cel-miR-39 was used as normalization control for all samples. Data presented as relative miRNA fold change. Each bar represents mean (SD) of three independent experiments (n=3).

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Figure 3.

miRNA spectra of the supernatants and the pellets obtained after ultracentrifugation of MCF7 cells conditioned media (A) and human blood plasma (B) as measured by TaqMan miRNA Low Density arrays (Applied Biosystems). Blood plasma (1ml) was combined with 11ml of 1×PBS before ultracentrifugation. ‘Left’: Correlation plots shows C_t values of miRNA signals obtained from total RNA extracted from 400μl (1/30 volume) of supernatants and total pellets (pellets were obtained after ultracentrifugation of total 12ml of samples). Note that only 1/30 of total supernatants were taken for the RNA isolation and subsequently for the TaqMan Low Density Arrays. MiRNAs which were undetected on the array (C_t>35) were assigned C_t values of 40. ES—miRNAs presented exclusively in the supernatant; EP—miRNAs presented exclusively in the pellets (exclusively detected miRNAs are listed in Supplementary Data). ‘Right’: Those miRNAs that were detected in both supernatants and pellets demonstrated a dramatic difference in the content. Note: amount of miRNAs in the pellets is much lower than in supernatants.

Extracellular miRNA is associated with Ago2 protein

We further hypothesized that exosome-free extracellular miRNA could be protected from nuclease digestion by associated proteins. To estimate the approximate molecular weight of ‘extracellular miRNA binding protein’, we applied a sequential ultrafiltration procedure (Figure 2A and B). For this purpose 0.22μm filtered blood plasma and conditioned media were further fractionated by centrifugal ultrafiltration using nanomembrane concentrators of 300, 100 and 50kDa MWCO. Individual TaqMan miRNA Assays were used to quantify the species of hsa-miR-16, hsa-miR-21 and hsa-miR-24 which remained in the samples after each ultrafiltration step. In conditioned media, extracellular miRNA completely passed through 300kDa MWCO filters, ~50% of the investigated miRNAs passed through 100kDa MWCO filter and only a negligible amount of miRNAs were found in the 50kDa filtrate (Figure 2C). The fact that 300kDa MWCO nanomembranes were permeable for extracellular miRNAs provided an additional evidence of exosome-free presence of extracellular miRNA. Similarly to the conditioned media, most of the miRNA was still found in the 300kDa filtrates in blood plasma (Figure 2D). However, roughly 90% of miRNA did not pass through 100kDa filters and 50kDa filtrates were almost totally depleted of miRNAs (Figure 2D). When serum-free 1-day conditioned media from MCF7 cells was sequentially fractionated, the percent of miRNA that passed through 100kDa MWCO nanomembranes was about 70% while near 5% of the original miRNA amount was still detected in 50kDa filtrate (Figure 4A). This data suggest that extracellular miRNA in blood plasma and conditioned media is associated with a protein of MW between 300 and 50kDa. It is well-known that in the cell mature miRNAs are directly bound to Ago2, a 96kDa protein responsible for the endonuclease cleavage of targeted mRNA (1). Accordingly, anti-Ago2 monoclonal antibody has previously been shown to be effective in capturing cellular miRNAs (19). To determine if the extracellular miRNA is also associated with Ago2 protein, the retinates (remnants that did not pass through the nanofilters) collected after ultrafiltration of serum-free conditional media (Figure 4A) were subjected to western immunoblot analysis with anti-Ago2 monoclonal antibody (Figure 4B). As we expected, Ago2 specific bands were observed in both 100 and 50kDa retinates, while no Ago2 was detected in 300kDa retinates. The relative intensities of Ago2 bands on the western blot corresponded to the amounts of miRNAs depleted from the filtrates (Figure 4A). More importantly, the amount of Ago2 in MCF7 conditioned media and in MCF7 cell lysates was proportional to their miRNAs content that was estimated by visual comparison of Ago2 signals on western blot and normalized C_t values of miR-16 and miR-24 (Figure 4C). To further confirm the binding of extracellular miRNAs to Ago2 protein in blood plasma and conditioned media, anti-Ago2 and control immunoprecipitates were subjected to TaqMan miRNA Assays (Figure 4D and E). In consistency with the western blot data, miRNAs were detected in anti-Ago2 immunoprecipitates while control immunoprecipitates (containing no anti-Ago2) were almost totally free of miRNAs. Moreover, incubation of both blood plasma and cells media with anti-Ago2 antibody caused the depletion of miRNA from the samples. Ultrafiltration and immunoprecipitation experiments were performed in a detergent-free environment, therefore, targeting a native form of extracellular miRNA/Ago2. Figure 4D and E included only the co-immunoprecipitation performed when 5μg of anti-Ago2 was used (when the maximal recovery of miRNAs was observed). Further increase of the anti-Ago2 antibody per reaction (from 5 to 10μg) did not significantly increase miRNA content in the pellet. The second immunoprecipitation with anti-Ago2 yielded significantly smaller amount of miRNA as compared to first immunoprecipitation (data not shown). The remaining miRNA in the supernatant is, therefore, unlikely to be attributed to larger parts to the efficiency of co-immunoprecipitation but could be associated with other Ago proteins, specifically Ago1, Ago3 and Ago4. Unfortunately, there are no well described antibodies against Ago1, Ago3 and Ago4 for the immunoprecipitation available. To test the hypothesis that other Ago proteins loaded with miRNA can also be present in the extracellular environment we have tested the ability of anti-FLAG antibody to precipitate miRNAs from 3-days conditional media of cells transfected with constructs encoding FLAG-fused human Ago1, Ago2, Ago3 and Ago4 (Figure 4F). The highest amount of miRNA in the media was still associated with Ago2; however, the media of the cells transfected with Ago1 also contained significant amounts of miRNA. It is therefore, feasible that the remaining miRNA in the blood plasma and conditioned media can be bound to other members of the Ago family.

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Figure 4.

(A–C) The levels of extracellular miRNAs correlate with the level of extracellular Ago2 but not NPM1 protein. One-day serum-free conditioned media from MCF7 cells was fractionated using nanomembrane concentrators of 300, 100 and 50kDa MWCO. (A) Individual TaqMan miRNA assays were used to quantify the species of miR-16, miR-21 and miR-24 which remained in the solution after each ultrafiltration step. (B) Concentrated fractions (retinates) were subjected to western immunoblot analysis using anti-Ago2 and anti-NPM1 antibody. Note: 300R=retinate from 300 kDa MWCO filter; 100R=100kDa retinate from 100kDa MWCO filter and 50R=retinate from 50kDa MWCO filter. (C) The amount of Ago2/miRNA in conditioned media was proportional to the amount of Ago2/miRNA in the cells. MCF7 cells lysate and concentrated serum-free conditioned media were prepared and miRNA content was assessed using TaqMan miRNA assays. After that, cell lysate was diluted so that the miRNA content in it equaled approximately the miRNA content in the concentrated serum-free media. Finally, western immunoblot was performed on concentrated media and diluted cell lysates and the amounts of Ago2 were visually compared. (D and E) Extracellular miRNA co-immunoprecipitated with anti-Ago2 antibody in a detergent-free environment. Species of miR-16, miR-21, miR-24 and exogenously expressed miR-34b were measured both in anti-Ago2 co-immunoprecipitates (P and P+Ago2) and in the remained supernatants (S and S+Ago2) obtained from MCF7 conditioned media (D) and blood plasma (E). Data presented as a fold change compared to control supernatants (assigned value 1), each bar represents mean (SD) of three independent experiments. Cel-miR-39 spiked in during RNA isolation step was used as normalization control. Note, the anti-Ago2 antibody induced a depletion of the miRNAs from conditioned media and blood plasma. (F) The levels of miR-16, miR-21 and miR-24 were measured in the anti-FLAG pellets obtained from HEK293T cells conditioned medias 72h after transfection with plasmids-encoding FLAG-Ago1, FLAG-Ago2, FLAG-Ago3, FLAG-Ago4 or empty vector (K).

In one recent report it was hypothesized that miRNA in serum-free conditioned media can be bound to nucleophosmin (NPM1) (13). This assumption was based on the fact that NPM1 was able to protect miRNA from RNAses when mixed with synthetic miRNA in vitro and was present in serum-free conditioned media in relatively high amounts. We indeed detected high amounts of NPM1 in 1-day conditioned serum-free media from MCF7 cells. However, the ultrafiltration patterns of NPM1 and extracellular miRNA were significantly different (Figure 4A and B). It remains to be elucidated, why 33kDa NPM1 was found exclusively in 100kDa retinate fraction (did not pass through 100kDa filters). However, in the cell NPM1 forms complexes with some other proteins (like nucleolin and ribonucleoproteins) and undergo oligomerization (20).

A.T. designed and carried out experimental part and wrote this manuscript; L.W. performed western blotting experiments and revised the manuscript; A.L. helped to establish miRNA isolation protocol and participated in qPCR experiments, B.B. conceived of the study, participated in its design and coordination and revised the manuscript.