67 Sample Preparation & Processing


Delivers Higher Concentration


Exosomes contain RNA of different sizes, producing BioAnalyzer broad peaks of between 20 and 30 nucleotides, suggesting a large microRNA (miRNA) population, with other peaks representative of messenger RNA (mRNA), ribosomal RNA (rRNA) and precursor RNA. While the RNA obtained from the crude exosomes and the density gradient- purifi ed exosomes were similar in size, it was noted that the concentration of the RNA obtained by density gradient ultracentrifugation was signifi cantly higher.


The RNA isolated from each exosomal sample was prepared into small RNA sequencing libraries, using a preparation kit on the Biomek workstation. The RNA from crude (ultracentrifugation only) and density gradient preparations from the HCT and CCD cell lines were prepared into small RNA libraries for NGS using a 50-cycle sequencing kit. The CCD 841 CoN and HCT 116 density gradient exosomal RNA generated 678,231 and 600,307 read counts, respectively, whereas CCD 841 CoN and HCT 116 crude exosomal RNA created 660,025 and 617,001 read counts, respectively. The high number of reads and low variation between data sets suggested that the isolated RNA is robust and of high enough yield for sequencing.


Variation and Upregulation


The authors’ results showed signifi cant variation in RNA type between cell lines and preparation methods (Figure 3). In the top plot, it is evident that the total relative abundance of miRNA is the lowest in the HCT 116 density gradient preparation method, but that mature miRNA is actually the greatest in this preparation (third plot). The greatest variation between preparation methods was evident with the HCT 116 cells. In terms of small RNA, signifi cantly more exons were present in the density gradient preparation, but this translated into additional GtRNA, long non-coding RNA, piRNA, precursor RNA, snoRNA and snRNA for the crude preparation. Of the abundant RNA, human rRNA was the most prevalent in all cell lines and preparation methods.


of results (Table 1). By combining automation with the benefi ts of density gradient ultracentrifugation, the results demonstrate a workfl ow capable of producing high-impact NGS data from a single sequencing run, which can be used to identify potential biomarkers and measure differential expression against sample type.


Table 1. Standardised exosome workfl ow.


Cell culture Viability assay Differential pelleting


Automated density gradient for highly pure isolation Particle characterisation Small RNA extraction NGS library construction Next-generation sequencing


For further information on Beckman Coulter centrifugation: beckman.com/centrifugation and http://info.beckmancoulter.com/ExosomeIsolation


*Collaborator systems and processes used included: Qiagen’s miRNeasy kits; Thermo Scientifi c NanoDrop 8000; Agilent BioAnalyzer Pico Chip/ BioAnalyzer 2100; Beckman Coulter Optima XPN ultracentrifuge with 45 Ti and SW32 Ti and SW41 Ti rotors; Allegra X-15 R with SX4750A rotor; Optima Max-XP benchtop centrifuge with TLA 120.2 rotor; Vi-Cell Viability Counter; DelsaMax Pro; Biomek 4000 Laboratory Automation Workstation; Greiner T-175 fl asks; Becton Dickinson cell culture plates; Quant-iT RiboGreen (Life Technologies) with SpectraMax i3 plate fl uorometer (Molecular Devices); NEBNext Small RNA Library Preparation Kit for Illumina (New England Biolabs); llumina MiSeq with 50 cycle single read/ Illumina BaseSpace Small RNA application.


References


1. Vader P, Breakefi eld XO, Wood MJ. Extracellular vesicles: emerging targets for cancer therapy. Trends Mol Med 2014; 20 (7): 385–93.


2. El Andaloussi S, Mager I, Breakefi eld XO, Wood MJ. Extracellular vesicles: biology and emerging therapeutic opportunities. Nat Rev Drug Discov 2013; 12 (5): 347–57.


3. Simpson RJ, Lim JW, Moritz RL, Mathivanan S. Exosomes: proteomic insights and diagnostic potential. Expert Rev Proteomics 2009; 6 (3): 267–83.


4. De Toro J, Herschlik L, Waldner C, Mongini C. Emerging roles of exosomes in normal and pathological conditions: new insights for diagnosis and therapeutic applications. Front Immunol 2015; 6: 203.


5. Amabile N, Rautou PE, Tedgui A, Boulanger CM. Microparticles: key protagonists in cardiovascular disorders. Semin Thromb Hemost 2010; 36 (8): 907–16.


6. DeJong OG, Verhaar MC, Chen Y et.al. Cellular stress conditions are refl ected in the protein and RNA content of endothelial cell-derived exosomes. J Extracell Vesicles 2012; 1: doi: 10.3402/jev. v1i0.18396.


7. Waldenström A, Gennebäck N, Hellman U, Ronquist G. Cardiomyocyte microvesicles contain DNA/ RNA and convey biological messages to target cells. PLoS One 2012; 7 (4): e34653.


8. Robbins PD, Morelli AE. Regulation of immune responses by extracellular vesicles. Nat Rev Immunol 2014; 14 (3): 195–208.


Figure 3. Relative abundance chart of RNA type following FASTQ generation and read-trimming.


Expression heat maps of the sequenced precursor and mature miRNA were also plotted to represent differential expression between preparation methods within a cell line. However, by further analysis, it was evident that many miRNA families also had signifi cant differential expression between cancer and normal colon cell lines. Fifteen gene families were determined to be signifi cantly differentially-expressed. Of note, mir-1246, mir-182, and mir-183 were all signifi cantly up-regulated (>3.75-fold change) in the colon cancer cell line CCD 841 CoN. In fact, these three gene families have previously been identifi ed to be upregulated in colon cancer [13, 16, 17], aligning well with the present authors’ results.


An Emerging Field


Standardisation of isolation and characterisation methods is critical to advances in this emerging fi eld. Density gradient ultracentrifugation is frequently the preferred choice for exosome isolation, generating pure sample preparations; however, the workfl ow often lacks reproducibility between laboratories and users. Next-generation small RNA sequencing is one of many downstream assays for exosome characterisation and biomarker identifi cation, but the protocols employed often vary drastically.


Here, the authors introduce a solution that uses a breadth of Beckman Coulter instrumentation to increase throughput, walkaway time, reproducibility, and accuracy


9. Rajendran L, Honsho M, Zahn TR et al. Alzheimer’s disease beta-amyloid peptides are released in association with exosomes. Proc Natl Acad Sci USA 2006; 103 (30): 11172–7.


10. Danzer KM, Kranich LR, Ruf WP et al. Exosomal cell-to-cell transmission of alpha synuclein oligomers. Mol Neurodegener 2012; 7: 42.


11. Kruh-Garcia NA, Wolfe LM, Chaisson LH et al. Detection of Mycobacterium tuberculosis peptides in the exosomes of patients with active and latent M. tuberculosis infection using MRM-MS. PLoS One 2014; 9 (7): e103811.


12. Colino J, Snapper CM. Exosomes from bone marrow dendritic cells pulsed with diphtheria toxoid preferentially induce type 1 antigen-specifi c IgG responses in naïve recipients in the absence of free antigen. J Immunol 2006; 177 (6): 3757–62.


13. Gould SJ, Booth AM, Hildreth JE. The Trojan exosome hypothesis. Proc Natl Acad Sci USA 2003; 100 (19): 10592–7.


14. Ogata-Kawata H, Izumiya M, Kurioka D et al. Circulating exosomal microRNAs as biomarkers of colon cancer. PLoS One 2014; 9 (4): e92921.


15. Swartz C, Smith Z. A standardized, automated approach for exosome isolation and characterization using Beckman Coulter instrumentation (http://info.beckmancoulter.com/ ExosomeIsolation).


16. Perilli L, Vicentini C, Agostini M et al. Circulating miR-182 is a biomarker of colorectal adenocarcinoma progression. Oncotarget 2014; 5 (16): 6611–9.


17. Zhou T, Zhang GJ, Zhou H, Xiao HX, Li Y. Overexpression of microRNA-183 in human colorectal cancer and its clinical signifi cance. Eur J Gastroenterol Hepatol 2014; 26 (2): 229–33.


Read, Share and Comment on this Article, visit: www.labmate-online.com/articles


Page 1  |  Page 2  |  Page 3  |  Page 4  |  Page 5  |  Page 6  |  Page 7  |  Page 8  |  Page 9  |  Page 10  |  Page 11  |  Page 12  |  Page 13  |  Page 14  |  Page 15  |  Page 16  |  Page 17  |  Page 18  |  Page 19  |  Page 20  |  Page 21  |  Page 22  |  Page 23  |  Page 24  |  Page 25  |  Page 26  |  Page 27  |  Page 28  |  Page 29  |  Page 30  |  Page 31  |  Page 32  |  Page 33  |  Page 34  |  Page 35  |  Page 36  |  Page 37  |  Page 38  |  Page 39  |  Page 40  |  Page 41  |  Page 42  |  Page 43  |  Page 44  |  Page 45  |  Page 46  |  Page 47  |  Page 48  |  Page 49  |  Page 50  |  Page 51  |  Page 52  |  Page 53  |  Page 54  |  Page 55  |  Page 56  |  Page 57  |  Page 58  |  Page 59  |  Page 60  |  Page 61  |  Page 62  |  Page 63  |  Page 64  |  Page 65  |  Page 66  |  Page 67  |  Page 68  |  Page 69  |  Page 70  |  Page 71  |  Page 72  |  Page 73  |  Page 74  |  Page 75  |  Page 76  |  Page 77  |  Page 78  |  Page 79  |  Page 80  |  Page 81  |  Page 82  |  Page 83  |  Page 84  |  Page 85  |  Page 86  |  Page 87  |  Page 88