Molecular Characterization of Peripheral Extracellular Vesicles in Clinically Isolated Syndrome: Preliminary Suggestions from a Pilot Study
Abstract
:1. Introduction
2. Materials and Methods
2.1. Study Population
2.2. Extracellular Vesicles Purification
2.3. Total RNA Isolation
2.4. Small RNA Deep Sequencing
2.5. Bioinformatic and Statistical Analysis of Small RNA Data
3. Results
4. Discussion
5. Take-Home Messages
- The analysis of EVs contents (especially sRNAs) may help to provide more information on their functional role(s) in several diseases; EVs contents may also be used for the isolation of circulating biomarkers (e.g., miRNAs).
- Few studies have investigated both the EV classes (Microvesicles and Exosomes) extracted simultaneously from the peripheral blood samples in the same patient, but this strategy should be suggested in order to fully understand the entire molecular picture of a given status.
- smallRNA deep sequencing is a valuable approach for studying the different classes of small non-coding RNA, although a solid bioinformatics support is recommended for the analysis and the interpretation of the obtained results.
- In order to obtain the right amount of evRNAs for the subsequent sRNA-seq analysis, we suggest/recommend to collect a higher quantity of blood samples (at least 20 mL/each) or to pool together samples belonging to different subjects with the same phenotype [34].
Supplementary Materials
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Microvesicles | Exosomes | |||||||||
---|---|---|---|---|---|---|---|---|---|---|
HC-1 | HC-2 | CIS-1 | HC-3 | CIS-2 | ||||||
Read Count | % | Read Count | % | Read Count | % | Read Count | % | Read Count | % | |
Raw reads | 12,031,509 | 26,023,784 | 23,176,535 | 57,568,091 | 17,246,086 | |||||
Clean reads (mapped/unmapped) | 3,851,795 | 18,679,042 | 7,845,182 | 39,163,534 | 10,030,669 | |||||
Mapped ncRNA | ||||||||||
miRNA | 73,459 | 1.91 | 143,322 | 0.77 | 1,382,492 | 17.62 | 829,102 | 2.12 | 91,160 | 0.91 |
miRNA_primary_transcript | 13,089 | 0.34 | 12,758 | 0.07 | 66,145 | 0.84 | 5,419,917 | 13.84 | 520,722 | 5.19 |
piRNA | 70,927 | 1.84 | 497,413 | 2.66 | 653,901 | 8.34 | 1,587,896 | 4.05 | 172,016 | 1.71 |
misc_RNA | 52,601 | 1.37 | 273,814 | 1.47 | 119,722 | 1.53 | 178,440 | 0.46 | 77,591 | 0.77 |
rRNA | 34,888 | 0.91 | 786,417 | 4.21 | 36,569 | 0.47 | 3,971,869 | 10.14 | 534,541 | 5.33 |
snoRNA | 754 | 0.02 | 1330 | 0.01 | 3442 | 0.04 | 72,452 | 0.18 | 15,955 | 0.16 |
snRNA | 2091 | 0.05 | 14,445 | 0.08 | 11,740 | 0.15 | 95,877 | 0.24 | 83,351 | 0.83 |
SRP_RNA | 2810 | 0.07 | 22,088 | 0.12 | 7666 | 0.10 | 10,246 | 0.03 | 829 | 0.01 |
tRNA | 12,925 | 0.34 | 465,531 | 2.49 | 42,955 | 0.55 | 276,975 | 0.71 | 23,337 | 0.23 |
vault_RNA | 106 | 0.001 | 2331 | 0.01 | 793 | 0.001 | 14,463 | 0.04 | 2120 | 0.02 |
Y_RNA | 27,020 | 0.70 | 276,914 | 1.48 | 454,862 | 5.80 | 390,174 | 1.00 | 31,963 | 0.32 |
antisense | 28,863 | 0.75 | 90,018 | 0.48 | 44,561 | 0.57 | 347,191 | 0.89 | 95,003 | 0.95 |
lincRNA | 573,422 | 14.89 | 215,484 | 1.15 | 338,285 | 4.31 | 1,227,887 | 3.14 | 1,289,945 | 12.86 |
lncRNA | 158,954 | 4.13 | 214,994 | 1.15 | 102,465 | 1.31 | 279,388 | 0.71 | 177,791 | 1.77 |
processed_transcript | 134,450 | 3.49 | 268,735 | 1.44 | 128,558 | 1.64 | 264,513 | 0.68 | 121,479 | 1.21 |
retained_intron | 102,548 | 2.66 | 180,506 | 0.97 | 126,455 | 1.61 | 530,026 | 1.35 | 228,149 | 2.27 |
Unmapped ncRNA | ||||||||||
Human genome (GRCh38) | 856,079 | 22.23 | 1,643,508 | 8.80 | 1,975,727 | 25.18 | 16,540,083 | 42.23 | 5,153,999 | 51.38 |
unmapped Human genome | 1,706,809 | 44.31 | 13,569,434 | 72.65 | 2,348,844 | 29.94 | 7,127,035 | 18.20 | 1,410,718 | 14.06 |
MVs | EXOs | ||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|
HCs-1-2 | CIS-1 | HC-3 | CIS-2 | ||||||||
Transcript ID | Read Count * | % | Transcript ID | Read Count | % | Transcript ID | Read Count | % | Transcript ID | Read Count | % |
hsa-miR-126-5p | 33,125.28 | 12.11 | hsa-miR-126-5p | 775,815 | 30.67 | hsa-miR-25-3p | 129,373 | 15.46 | hsa-let-7a-5p | 24,658 | 10.88 |
hsa-let-7f-5p | 26,785.99 | 9.80 | hsa-let-7f-5p | 341,213 | 13.49 | hsa-let-7b-5p | 60,980 | 7.29 | hsa-miR-25-3p | 15,906 | 7.02 |
hsa-let-7a-5p | 26,706.14 | 9.77 | hsa-let-7a-5p | 266,308 | 10.53 | hsa-miR-451a | 60,215 | 7.20 | hsa-miR-3908 | 15,510 | 6.84 |
hsa-miR-142-3p | 16,230.72 | 5.94 | hsa-miR-23a-3p | 199,088 | 7.87 | hsa-let-7a-5p | 51,475 | 6.15 | hsa-let-7f-5p | 13,026 | 5.75 |
hsa-miR-1246 | 15,182.89 | 5.55 | hsa-miR-223-3p | 172,699 | 6.83 | hsa-let-7g-5p | 38,560 | 4.61 | hsa-miR-126-5p | 11,069 | 4.88 |
hsa-miR-126-3p | 11,081.05 | 4.05 | hsa-miR-150-5p | 113,228 | 4.48 | hsa-miR-19b-3p | 28,400 | 3.39 | hsa-miR-5096 | 10,094 | 4.45 |
hsa-miR-23a-3p | 10,809.96 | 3.95 | hsa-miR-151a-3p | 34,022 | 1.35 | hsa-miR-24-3p | 28,108 | 3.36 | hsa-let-7b-5p | 8506 | 3.75 |
hsa-miR-223-3p | 10,415.68 | 3.81 | hsa-let-7b-5p | 33,279 | 1.32 | hsa-miR-1246 | 26,006 | 3.11 | hsa-miR-1273c | 7323 | 3.23 |
hsa-miR-150-5p | 10,384.42 | 3.80 | hsa-let-7g-5p | 32,259 | 1.28 | hsa-miR-126-5p | 24,775 | 2.96 | hsa-miR-8086 | 6988 | 3.08 |
hsa-miR-1260b | 8540.57 | 3.12 | hsa-miR-146a-5p | 26,769 | 1.06 | hsa-miR-122-5p | 24,611 | 2.94 | hsa-let-7g-5p | 6770 | 2.99 |
hsa-let-7b-5p | 7040.47 | 2.57 | hsa-miR-25-3p | 26,213 | 1.04 | hsa-let-7f-5p | 24,091 | 2.88 | hsa-miR-23a-3p | 6519 | 2.88 |
hsa-miR-191-5p | 5648.99 | 2.07 | hsa-miR-24-3p | 23,816 | 0.94 | hsa-miR-23a-3p | 23,673 | 2.83 | hsa-miR-1290 | 6292 | 2.78 |
hsa-miR-24-3p | 5522.51 | 2.02 | hsa-miR-1260b | 23,499 | 0.93 | hsa-miR-486-5p | 23,461 | 2.80 | hsa-miR-7704 | 5136 | 2.27 |
hsa-let-7g-5p | 3832.22 | 1.40 | hsa-miR-126-3p | 22,682 | 0.90 | hsa-miR-92a-3p | 21,203 | 2.53 | hsa-miR-223-3p | 4713 | 2.08 |
hsa-miR-25-3p | 3070.25 | 1.12 | hsa-miR-21-5p | 21,229 | 0.84 | hsa-miR-223-3p | 15,895 | 1.90 | hsa-miR-1303 | 3914 | 1.73 |
hsa-miR-146a-5p | 2287.37 | 0.84 | hsa-miR-191-5p | 20,705 | 0.82 | hsa-miR-486-3p | 13,892 | 1.66 | hsa-miR-4279 | 3488 | 1.54 |
hsa-miR-92a-3p | 2115.47 | 0.77 | hsa-miR-92a-3p | 18,397 | 0.73 | hsa-miR-16-5p | 13,286 | 1.59 | hsa-miR-6087 | 3312 | 1.46 |
hsa-miR-151a-3p | 1937.60 | 0.71 | hsa-let-7d-3p | 17,960 | 0.71 | hsa-miR-191-5p | 12,984 | 1.55 | hsa-miR-19b-3p | 3103 | 1.37 |
hsa-miR-342-3p | 1760.54 | 0.64 | hsa-miR-23b-3p | 14,808 | 0.59 | hsa-miR-93-5p | 12,851 | 1.54 | hsa-miR-24-3p | 2979 | 1.31 |
hsa-miR-486-3p | 1706.72 | 0.62 | hsa-miR-374b-5p | 12,218 | 0.48 | hsa-miR-19a-3p | 11,956 | 1.43 | hsa-miR-6087 | 2622 | 1.16 |
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Nuzziello, N.; Blonda, M.; Licciulli, F.; Liuni, S.; Amoruso, A.; Valletti, A.; Consiglio, A.; Avolio, C.; Liguori, M. Molecular Characterization of Peripheral Extracellular Vesicles in Clinically Isolated Syndrome: Preliminary Suggestions from a Pilot Study. Med. Sci. 2017, 5, 19. https://doi.org/10.3390/medsci5030019
Nuzziello N, Blonda M, Licciulli F, Liuni S, Amoruso A, Valletti A, Consiglio A, Avolio C, Liguori M. Molecular Characterization of Peripheral Extracellular Vesicles in Clinically Isolated Syndrome: Preliminary Suggestions from a Pilot Study. Medical Sciences. 2017; 5(3):19. https://doi.org/10.3390/medsci5030019
Chicago/Turabian StyleNuzziello, Nicoletta, Maria Blonda, Flavio Licciulli, Sabino Liuni, Antonella Amoruso, Alessio Valletti, Arianna Consiglio, Carlo Avolio, and Maria Liguori. 2017. "Molecular Characterization of Peripheral Extracellular Vesicles in Clinically Isolated Syndrome: Preliminary Suggestions from a Pilot Study" Medical Sciences 5, no. 3: 19. https://doi.org/10.3390/medsci5030019