Extracellular vesicles are composed of a lipid bilayer that is produced by various cell types. These vesicles are primarily categorized by mode of biogenesis into 3 classes: exosomes, microvesicles, and apoptotic bodies. Exosomes are formed as the intraluminal vesicles of multivesicular bodies during the maturation of endosomes and are secreted from cells by exocytosis after the multivesicular bodies fuse with a plasma membrane.1 Microvesicles and apoptotic bodies form by direct budding from the plasma membrane.1 The size distributions of exosomes and microvesicles overlap; therefore, size alone cannot be used to distinguish between the 2.2 And because no standards have been established to separate or classify the various vesicle types, the collective term EV is recommended.
Extracellular vesicles have been detected in various mammalian body fluids, including blood, CSF, saliva, amniotic fluid, breast milk, and urine.3–7 These microstructures package nucleic acids, proteins, and lipids in lipid bilayers and function in cell-to-cell communication by shuttling complex messages.
Most extracellular RNAs are encased within membranous vesicles or are tightly associated with proteins to avoid degradation.8 The miRNA is a small noncoding molecule that has been implicated in posttranscriptional gene regulatory mechanisms.9 Studies10–13 have shown that the patterns of miRNAs in EVs may reflect certain pathophysiologic conditions in humans. Extracellular miRNAs are also associated with immunosuppression of antitumor responses, tumor growth, metastatic progression, neuronal signaling pathways, and transmission of drug resistance to sensitive cells.14–16 Therefore, miRNAs in EVs may be novel candidates for the detection and prediction of various diseases, such as cancer, neurodegenerative diseases, and immune disorders.17–19
Several methods are used to collect EVs, including membrane affinity chromatography, use of antibody-coated magnetic beads, filtration technologies, microfluidics, precipitation techniques, and ultracentrifugation.20,21 For clinical applications in dogs, miRNAs need to be stably harvested from a small amount of sample. Despite recent advances in our biological understanding of EVs, a lack of standardized methods exists for isolating EVs from human and canine plasma samples.
Suitable reference genes are also needed to accurately compare miRNA profiles between the target disease and other diseases. Exogenous (external) reference genes (referred to as spike-in genes) and endogenous (internal) reference genes (also referred to as housekeeping genes) have recently been used to normalize miRNA expression data. The spike-in method can eliminate some deviations related to the experimental process but does not correct for deviations in collection methods and quality of EV samples.22 Although endogenous reference genes are also needed to normalize miRNA expression data, no such genes have been established for plasma EVs in dogs. The purpose of the study reported here was to compare ultracentrifugation, precipitation, and membrane affinity chromatography methods for the isolation of EVs from canine plasma samples and to identify suitable endogenous reference genes for incorporation into a qRT-PCR assay of miRNA expression in these EVs.
Materials and Methods
Animals and sample collection
Six healthy Beagles (4 sexually intact females and 2 castrated males) from an academic research facility were included in the study. Median age was 2 years (range, 1 to 4 years). One blood sample (2 mL) was collected from a jugular or cephalic vein in each dog via 23-gauge needles into 2-mL EDTA-containing evacuated tubes.a Samples were centrifuged at 800 × g for 20 minutes, and the supernatant (plasma) was harvested and stored at −80°C. All procedures were approved by the Institutional Animal Care and Use Committee of Gifu University.
Isolation of EVs from plasma samples
Plasma samples were thawed in a thermostatic water bath at 37°C, and EVs were isolated from thawed samples by means of 3 methods: ultracentrifugation, precipitation, and membrane affinity chromatography. For the ultracentrifugation method, 0.3 mL of plasma was mixed with 1.2 mL of PBS solution. The mixture was ultracentrifuged by use of a swinging bucket rotorb at 100,000 × g and 4°C for 1 hour.20,23 The resulting pellet was resuspended in 0.3 mL of nuclease-free water. For the precipitation method, commercial kitsc were used in accordance with the manufacturer's instructions. Briefly, 0.3 mL of plasma was mixed with thrombin and incubated for 5 minutes at room temperature (approx 25°C). The mixture was centrifuged at 10,000 × g for 5 minutes, and the supernatant was collected. Collected supernatant was mixed with the precipitation buffer and incubated for 1 hour at 4°C. The mixture was centrifuged at 500 × g for 5 minutes, and the pellet was resuspended. For the membrane affinity chromatography method, commercial kitsd were used in accordance with the manufacturer's instructions. Briefly, 0.3 mL of plasma was mixed with binding buffer and added to the membrane affinity column. The column was centrifuged at 500 × g for 1 minute, then washed with buffer, and the EVs were eluted.
Assay of EVs with nanoparticle tracking analysis
The concentration and size distribution of EVs isolated by the ultracentrifugation method (and no other method) were measured by nanoparticle tracking analysis in accordance with the instructions provided with the nanoparticle tracking instrument.e Temperature was monitored throughout measurements. Briefly, EVs isolated by ultracentrifugation were homogenized by vortexing and then diluted with PBS solution at a ratio of 1:100. A camera level of 12 and automatic functions for all postacquisition settings were used for recording of the results, with the detection threshold optimized for samples. Polystyrene latex standards were analyzed to validate operation of the assay instrument. Five analyses were performed for each sample, and the mean of the 5 results was calculated.
Total RNA extraction and qRT-PCR assay
Total RNA was extracted from EVs collected by use of all 3 methods with a commercial kitf in accordance with the manufacturer's instructions. The miRNAs were subsequently reverse transcribed to cDNA by use of a commercial miRNA reverse transcription kit.g Expression of 4 miRNAs, including let-7a,h miR-16,i miR-26a,j and miR-103k examined as candidate reference genes, was assessed in duplicate by qRT-PCR assay with the use of custom-designed primersl (Appendix). The following thermal cyclerm conditions were used for the qRT-PCR assay: 1 cycle at 95°C for 10 minutes, 60 cycles of amplification at 95°C for 5 seconds, and 60°C for 30 seconds.
After the reactions had been completed, the Ct values, used as an indicator of the amount of miRNA expression (ie, expression level), were calculated by use of a manually set cycle threshold. At a level of 10 raw fluorescent units, all assayed plates fell within the exponential phases of the qRT-PCR reactions. Results of each duplicate assay were averaged to yield a mean Ct value.
Statistical analysis
Expression levels (Ct values) of each candidate reference gene were compared between pairs of isolation methods by means of 1-way ANOVA, followed by the Tukey test for multiple comparisons.n Values of P < 0.05 were considered significant.
The expression stability of candidate reference genes was calculated by use of 3 widely used computer algorithms, referred to in this report as A,24,o B,25,p and C.26,q Algorithm Ao calculated the pairwise variation in expression of 1 candidate reference gene relative to that of all other candidate reference genes as the SD of the logarithmically transformed expression (Ct) ratios. The candidate gene stability measure, M, was then calculated as the mean of these pairwise variations,24 with a value < 1.5 interpreted as indicating a stably expressed gene. Algorithm Bp calculated a stability value by use of the combined estimation of the variation in expression of each candidate gene within and between collection methods. Because this algorithm was not designed to use Ct values, Ct values were converted to a linear scale for this analysis.26
Algorithm Cq generated an index based on the geometric mean of all the candidate gene Ct values and determined the stability of genes on the basis of repeated pairwise correlations with one another and with the index.27 After the statistics for individual candidate genes were computed, the expression levels of the candidate reference genes were determined by inspecting calculated variations in SD values. According to the variability observed, reference genes were ordered from most stable (ie, with the lowest variation) to least stable (ie, with the highest variation).27 An SD value of < 1.0 was considered stable.
For each algorithm, candidate reference genes were ranked by stability. Then, for each gene, the geometric mean of the rank obtained by each of the 3 algorithms was calculated, and genes were reranked by the geometric mean values. The gene with the lowest geometric mean value was considered the most stable reference gene.28
Results
The Ct values of the 4 candidate reference genes (miRNAs) were compared among the 3 EV isolation methods (Figure 1). Results indicated that the miRNA expression levels in EVs isolated by the ultracentrifugation method had lower SDs and lower Ct values than in EVs isolated by other methods, suggesting that the ultracentrifugation method was the most stable for isolating miRNAs from plasma EVs. Assays of peak fractions with nanoparticle tracking revealed that EV samples obtained with the ultracentrifugation method contained a mean ± SD of approximately 1.59 × 1010 vesicles/mL ± 4.2 × 108 vesicles/mL. Mean ± SD size of the vesicles was 215 ± 4.0 nm (Figure 2).
Box plots of miRNA expression in EVs isolated from canine plasma samples (n = 6 dogs) by ultracentrifugation (method 1), precipitation (method 2), and membrane affinity chromatography (method 3). The central horizontal line within each box represents the median; the top and bottom of each box represent the upper and lower quartiles, respectively; and the whiskers represent the range. Dots represent single values. Values indicated by the tails of brackets are significantly (*P < 0.01; åP < 0.001; ‡P < 0.05) different as determined by 1-way ANOVA.
Citation: American Journal of Veterinary Research 80, 5; 10.2460/ajvr.80.5.449
Box plots of miRNA expression in EVs isolated from canine plasma samples (n = 6 dogs) by ultracentrifugation (method 1), precipitation (method 2), and membrane affinity chromatography (method 3). The central horizontal line within each box represents the median; the top and bottom of each box represent the upper and lower quartiles, respectively; and the whiskers represent the range. Dots represent single values. Values indicated by the tails of brackets are significantly (*P < 0.01; åP < 0.001; ‡P < 0.05) different as determined by 1-way ANOVA.
Citation: American Journal of Veterinary Research 80, 5; 10.2460/ajvr.80.5.449
Box plots of miRNA expression in EVs isolated from canine plasma samples (n = 6 dogs) by ultracentrifugation (method 1), precipitation (method 2), and membrane affinity chromatography (method 3). The central horizontal line within each box represents the median; the top and bottom of each box represent the upper and lower quartiles, respectively; and the whiskers represent the range. Dots represent single values. Values indicated by the tails of brackets are significantly (*P < 0.01; åP < 0.001; ‡P < 0.05) different as determined by 1-way ANOVA.
Citation: American Journal of Veterinary Research 80, 5; 10.2460/ajvr.80.5.449
Size distribution of EVs isolated from canine plasma samples (n = 1 dog) by ultracentrifugation as determined by nanoparticle tracking analysis. The line represents the mean concentration of EVs over the various sizes in 5 sets of analyses, and the gray-shaded area represents the SEM. Mean size at each concentration peak is indicated.
Citation: American Journal of Veterinary Research 80, 5; 10.2460/ajvr.80.5.449
Size distribution of EVs isolated from canine plasma samples (n = 1 dog) by ultracentrifugation as determined by nanoparticle tracking analysis. The line represents the mean concentration of EVs over the various sizes in 5 sets of analyses, and the gray-shaded area represents the SEM. Mean size at each concentration peak is indicated.
Citation: American Journal of Veterinary Research 80, 5; 10.2460/ajvr.80.5.449
Size distribution of EVs isolated from canine plasma samples (n = 1 dog) by ultracentrifugation as determined by nanoparticle tracking analysis. The line represents the mean concentration of EVs over the various sizes in 5 sets of analyses, and the gray-shaded area represents the SEM. Mean size at each concentration peak is indicated.
Citation: American Journal of Veterinary Research 80, 5; 10.2460/ajvr.80.5.449
Algorithm A revealed overall stability in expression for all 4 candidate reference genes. When the ultracentrifugation method was used for EV isolation, the most stable reference genes were let-7a and miR-103 (Table 1). Stability values for all 4 miRNAs were slightly larger (indicating less stability) than values obtained with the precipitation and membrane affinity chromatography methods. Algorithm B identified miR-103 as the most stably expressed reference gene when the ultracentrifugation method was used. Algorithm C revealed that all candidate reference genes had sufficient stability and that when the ultracentrifugation method was used, the most stable reference gene was miR-16. The geometric means of stability values indicated that, for all 3 algorithms, the candidate reference genes with the greatest stability were miR-103 when the ultracentrifugation method was used, miR-26a when the precipitation method was used, and miR-16 when the membrane affinity chromatography method was used.
Expression stability values and ranks of those values for 4 miRNAs in EVs isolated from canine plasma samples (n = 6) by use of 3 methods, as calculated by 3 algorithms.
| miRNA, by isolation method | Algorithm Ao | Algorithm Bp | Algorithm Cq | Geometric mean rank | Overall rank |
|---|---|---|---|---|---|
| Ultracentrifugation | |||||
| let-7a | 0.015 (1) | 0.873 (3) | 0.390 (2) | 1.82 | 2 |
| miR-16 | 0.021 (3) | 1.192 (4) | 0.340 (1) | 2.29 | 3 |
| miR-26a | 0.028 (4) | 0.720 (2) | 0.740 (4) | 3.17 | 4 |
| miR-103 | 0.015 (1) | 0.303 (1) | 0.430 (3) | 1.44 | 1 |
| Precipitation | |||||
| let-7a | 0.117 (4) | 0.426 (1) | 0.129 (4) | 2.52 | 3 |
| miR-16 | 0.044 (3) | 2.931 (4) | 0.025 (3) | 3.30 | 4 |
| miR-26a | 0.030 (1) | 0.847 (3) | 0.020 (1) | 1.44 | 1 |
| miR-103 | 0.030 (1) | 0.577 (2) | 0.022 (2) | 1.59 | 2 |
| Membrane affinity chromatography | |||||
| let-7a | 0.192 (1) | 0.415 (3) | 0.113 (1) | 1.44 | 2 |
| miR-16 | 0.192 (1) | 0.262 (1) | 0.113 (1) | 1 | 1 |
| miR-26a | 0.225 (4) | 1.442 (4) | 0.126 (3) | 3.63 | 4 |
| miR-103 | 0.218 (3) | 0.324 (2) | 0.115 (2) | 2.29 | 3 |
Values in parentheses represent ranks as indicated by the associated algorithm.
Discussion
In the study reported here, we extracted miRNAs from EVs that had been isolated from a small amount (0.3 mL) of plasma from healthy dogs. Results indicated that the ultracentrifugation method yielded the most abundant EV miRNAs and lowest variation among samples. Results also suggested that expression of miRNAs obtained by the precipitation method was less than that for miRNAs obtained by the ultracentrifugation method and markedly differed among the samples. Research in humans has shown that the precipitation method yields a higher amount of miRNAs than the ultracentrifugation method for exosomes from serum and CSF samples.29 This discrepancy in findings between studies may be due to the small volume of samples, type of samples (plasma vs serum), and differences in sample components.
The precipitation method allows precipitation of exosomes and other EVs.20 This method is based on capturing water molecules, which otherwise form the hydrate envelope of particles in suspension.30 Samples of EVs isolated by the ultracentrifugation and precipitation methods reportedly contain many nonvesicular contaminants, including protein aggregates and lipoproteins.20,31 These methods would precipitate miRNAs of both EVs and other sources, with the impurities affecting the reaction and, potentially, results for miRNA expression. The chromatography method involves affinity spin columns to isolate exosomes and other EVs.21 The amounts of target RNAs obtained from 4 mL of human plasma by the membrane affinity chromatography method are reportedly equal to those obtained by the ultracentrifugation method.32 Sample volume may be important for detecting stable reference and target genes for clinical applications.
Several endogenous reference genes for EV miRNAs exist in humans; however, none of these are universal for different tissue types and pathological conditions. The candidate reference genes evaluated in the present study, including the miRNAs let-7a, miR-16, miR-26a, and miR-103, were selected because they have been commonly used as the endogenous control genes for circulating EVs in humans.22,28,33 The miRNA miR-16 has been efficiently used as an endogenous reference gene in EVs from peritoneal lavage fluid and serum samples; however, it is reportedly unstable in serum-derived EVs from humans with hepatitis B or hepatocellular carcinoma and in those from healthy subjects.33–35 The stabilities of reference genes differed among EV isolation methods in the present study. Our results indicated that all evaluated miRNAs could be used as reference genes, whereas miR-103 was the most stable for normalizing expression values for miRNAs in EVs isolated from plasma samples of healthy dogs by the ultracentrifugation method.
Nanoparticle tracking analysis was used in the present study because it is accurate for sizing monodisperse and polydisperse samples and has a markedly better peak resolution than dynamic light scattering.36 The size distribution and number of EVs were determined only for those isolated by the ultracentrifugation method. Previous research has shown that serum EVs isolated by the ultracentrifugation method have a greater diameter than those isolated by the precipitation method.37 The nanoparticle tracking analysis was unable to discriminate different types of EVs in the present study. Because the size distribution of exosomes and microvesicles overlaps, size alone cannot be used to distinguish EV types.2 Additional research is needed to characterize isolated EVs, such as immunoblotting, mass spectrometry, and electron microscopy.
Plasma samples were selected for evaluation in the study reported here because the coagulation process affects the pattern of miRNAs in blood. The concentration of miRNAs is reportedly higher in serum samples than in corresponding plasma samples owing to the release of additional RNAs from RBCs and platelets during the coagulation process.38 Therefore, whether serum or plasma is to be used should be considered before attempting to characterize or quantify miRNAs of EVs in blood.
A limitation of the present study was the lack of analysis of the number of EVs isolated by all 3 methods; therefore, it was unclear whether all methods yielded EVs. Only 4 candidate reference genes were assessed by qRT-PCR assay, and additional research is needed to investigate the usefulness of other reference genes found in EVs. Another limitation was that the stabilities of the candidate reference genes may be affected by age, breed, and pathological conditions; therefore, additional studies are needed to identify stable reference genes in different conditions.
Acknowledgments
This study was performed at the Joint Department of Veterinary Medicine, Faculty of Applied Biological Sciences, Gifu University.
Funded in part by a Grant-in-Aid for Young Scientists (grant No. 17K15378) from the Japan Society for the Promotion of Sciences and a research grant from Gifu University.
The authors thank Yukina Kuwahara for technical assistance.
ABBREVIATIONS
| Ct | Threshold cycle |
| EV | Extracellular vesicle |
| miRNA | MicroRNA |
| qRT | Quantitative reverse transcription |
Footnotes
Venoject II, VP-NA050K, Terumo Corp, Tokyo, Japan.
S52ST rotor, Hitachi Inc, Tokyo, Japan.
miRCURY exosome isolation kit, Qiagen Inc, Hilden, Germany.
exoEasy Maxi kit, Qiagen Incorp, Hilden, Germany.
NanoSight LM10V-HS, Malvern Panalytical Ltd, Malvern, England.
NucleoSpin miRNA plasma kit, Machery-Nagel GmbH, Düren, Germany.
TaqMan MiRNA reverse transcription kit, ThermoFisher Scientific, Waltham, Mass.
cfa-let-7a assay (No. 000377), ThermoFisher Scientific, Waltham, Mass.
cfa-miR-16 assay (No. 000391), ThermoFisher Scientific, Waltham, Mass.
cfa-miR-26a assay (No. 000405), ThermoFisher Scientific, Waltham, Mass.
cfa-miR-103 assay (No. 000439), ThermoFisher Scientific, Waltham, Mass.
TaqMan small RNA assays, ThermoFisher Scientific, Waltham, Mass.
PCR thermal cycler Dice TP800, Takara Bio Incorp, Shiga, Japan.
JMP, version 13.2, SAS Institute Inc, Cary, NC.
geNorm, version 3.5, Center for Medical Genetics, Ghent, Belgium. Available at: genorm.cmgg.be. Accessed Nov 10, 2017.
NormFinder Excel add-in, version 0.953. Available at: moma.dk/normfinder-software. Accessed Jul 2, 2018.
BestKeeper, version 1. Available at: www.gene-quantification.de/bestkeeper.html. Accessed Nov 10, 2017.
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Appendix
List of Canine familiaris miRNA sequences for candidate reference genes in EVs isolated from canine plasma samples.
| miRNA | Sequence |
|---|---|
| let-7a-1 | 5′-UGAGGUAGUAGGUUGUAUAGUU-3′ |
| miR-16 | 5′-UAGCAGCACGUAAAUAUUGGCG-3′ |
| miR-26a | 5′-UUCAAGUAAUCCAGGAUAGGCU-3′ |
| miR-103 | 5′-AGCAGCAUUGUACAGGGCUAUGA-3′ |
