Fig 1.
Workflow for isolating RNA from extracellular vesicles using membrane affinity columns.
EV RNA is isolated from whole blood by separating the plasma or serum, pre-filtering the sample to exclude cell-contamination, and loading on the membrane affinity column followed by a brief wash. The bound vesicles are lysed and eluted with QIAzol; the RNA extracted by addition of chloroform, precipitated by ethanol and further purified using an RNeasy column.
Fig 2.
Scanning electron microscopy and western blot analysis of intact vesicles isolated by membrane affinity capture and ultracentrifugation.
(A) Scanning electron microscopy (SEM; 20.000 x magnification) of a solubilized pellet from ultracentrifugation of pre-filtered (0.8 μm) plasma compared to a non-lysed eluate from the membrane affinity spin column. Both preparations contain vesicle-shaped structures with an expected size range from 50–200 nm (white arrows; scale bar 200 nm) indicating that intact vesicles are eluted from the spin column membrane. (B) Exosomes were isolated from four milliliters of normal human plasma using either the membrane affinity column (lane 2) or ultracentrifugation (UC) method (lane 3). Exosomes were concentrated, washed, then lysed, and exosome protein lysates were processed as described in Materials and Methods. The signal for TSG101 runs close to the predicted molecular weight of 43 kDa, the specificity of the TSG101 antibody was confirmed by positive control HeLa cell lysates and further verified by the absence of the 46 kDa band when probed with secondary antibody only. The blot shown is a representative of at least three separate experiments, indicating that the exosome-enriched protein TSG101 is present in vesicles eluted from the membrane affinity column.
Fig 3.
NanoSight example data of vesicles eluted from the membrane affinity column and the corresponding flow-through.
Vesicles from 4 mL plasma were bound to a membrane affinity column, eluted from the column, and diluted into the working concentration of the NanoSight instrument. The flow-through from the membrane affinity column was kept and processed side by side with the eluate to compare both samples. Shown is the frequency and accumulated fractions of particle size (nm) in both samples types, differing in their average size (peak top noted in picture). While the majority of small protein complexes remains in the flow-through, leading to a small peak size of 99 nm, the eluate from the column contains vesicles, resulting in a larger peak size of 174 nm. The complete data for particle sizes and relative abundance of particles of different fractions from ultracentrifugation and membrane affinity elution is listed in Table 1 as mean and standard deviation (SD).
Table 1.
NanoSight data comparing particles in whole plasma with fractions from membrane affinity columns and ultracentrifugation (mean ± SD).
The values obtained for plasma and the column flow-through represent two replicate isolations, the values for the column eluate and the ultracentrifugation pellet represent four replicate isolations, and the values for the UC supernatant were measured with a single isolation replicate.
Fig 4.
Relative quantification of column-bound RNA after treatment with RNase and/or detergent.
Vesicles from 4 mL of pre-filtered plasma were bound to a membrane affinity column and washed. The column was treated for 30 minutes with either RNase A, the detergent Triton X-100, both, or reaction buffer only (mock-treatment). Subsequently, the RNA was isolated and analyzed using RT-qPCR against two mRNAs (GAPDH, HPRT1) and two miRNAs (miR-16, let-7a). The bar plots represent the relative amount of nucleic acids in the sample, compared to mock treatment alone, with columns as mean and whiskers as SD of two replicate isolations each. Assuming a perfect amplification efficiency, the % of PCR signal from mock is calculated as (2^(CT control–CT sample))*100 (see methods). Only when a detergent is used to destabilize the lipid-bilayers, the RNase is able to digest the RNA (leftmost columns), indicating that the procedure isolates membrane-protected RNA, a general property of EVs.
Fig 5.
Size distribution of total RNA from cancer patient plasma isolated by membrane affinity columns and ultracentrifugation.
Bioanalyzer sizing of vesicle-derived RNA purified by two methods. Total EV RNA from 2 mL plasma of a melanoma patient was isolated using membrane affinity columns and compared total EV RNA from ultracentrifugation, the current gold standard of EV isolation. Both methods purify RNA of similar size and yield.
Fig 6.
Reverse transcription of isolated total RNA using random primers and oligo-dT.
RNA from 2 mL of pre-filtered plasma was extracted with membrane affinity columns and reverse transcribed by superscript II using either oligo-dT 20mers, for priming of transcripts with intact poly-A tails, or random hexamers, for priming of both degraded and non-degraded transcripts. The scatterplot shows mean and SD of 6 independent RT reactions measured by 2 qPCR replicates for each of the two datasets. The distance of each RT-qPCR assay to the poly-A tail of the transcript are noted next to the plot. A good correlation of the raw CT values from random and oligo-dT priming demonstrates that the vast majority of assayed EV transcripts are intact and full-length.
Fig 7.
Recovery of mRNAs and miRNAs from plasma.
EV RNA from 0.2 mL of pre-filtered plasma was isolated using a membrane affinity column and RNA from the flow-through of the spin column was extracted using direct lysis with the miRNeasy Serum/Plasma kit. Shown are raw CT values from RT-qPCRs with rows as replicate isolations and colored diamonds as replicate qPCRs. Comparing the two fractions shows that the membrane affinity columns capture almost all mRNA and vesicle-specific miRNAs in plasma.
Fig 8.
Quantification of RNA yield with increasing plasma volumes.
EV RNA from increasing plasma volumes was extracted with membrane affinity columns and analyzed using RT-qPCR against two mRNAs (BRAF, HPRT1) and a miRNA known to be present in vesicles (let-7a). Shown are raw CT values with rows as individual extractions and colored diamonds as replicate qPCRs. The signal increase of 1 CT with each doubling of input amount into extraction demonstrates a linear efficiency of EV extraction up to volumes of 4 mL plasma.
Fig 9.
RNA extraction from high volumes of plasma and serum using several available commercial kits.
EV RNA from 4 mL of plasma or serum was isolated using membrane affinity (spin column), ultracentrifugation (Ultra) and three commercially available methods based on filtration (Kit B) or polymer-based precipitation (Kit S, Kit I) according to the manufacturers recommendations. The method marked with a star had no procedure for processing of high volumes available. The plot depicts raw CT values of 5 different individuals using an RT-qPCR assay against the GAPDH mRNA. Only column-based purification and ultracentrifugation efficiently recover RNA from high sample volumes.
Fig 10.
Detection of cancer-related genes in EV RNA from high volumes of plasma.
EV RNA from various volumes of plasma was isolated with membrane affinity columns, reverse transcribed and pre-amplified using the RT2 PCR system and detected using the Human Cancer Pathway Finder PCR Array. The boxplots depict the median and interquartile range of the obtained CT values. At the lowest input volume of 0.2 mL only 46% of the mRNAs are robustly detected (CT<30) but the isolation of higher volumes leads to a linear increase in CT signal and a corresponding rate of mRNA detection. Most of the assayed oncogenes are readily detected in plasma volumes equal or higher 2mL.