This work was funded by University of Helsinki project funding (WBS490302, WBS73714112) Helsinki University Hospital State funding for university-level health research (Y1014SUL05, TYH2016130), Finnish-Norwegian Medical Foundation, and the Selma and Maja-Lisa Selander Fund (Minerva Foundation). We thank Walter Pavicic for providing the modified MSPA protocol and for related technical support. We are grateful to Teemu Masalin (University of Helsinki) for helping us with video production.

This study was approved by the Ethics Committee of Helsinki and Uusimaa Hospital District (ethical approval D. No. 217/13/03/02/2015).
1. Preparation of EV-depleted FBS by ultracentrifugation
<ol>
	<li>Take FBS in (ultra)centrifuge tubes and place them in ultracentrifuge buckets. To ensure that the ultracentrifugation runs smoothly and safely, balance the buckets within 10 mg of each other.</li>
	<li>Load the buckets on a swinging rotor (type SW28, k-factor 246). Place the rotor in the ultra...

<ol>
	<li>Esteller, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+epigenomics:+DNA+methylomes+and+histone-modification+maps.">Cancer epigenomics: DNA methylomes and histone-modification maps.</a> Nature Reviews Genetics. 8, 286-298 (2007).</li><li>Herman, J. G., Baylin, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+silencing+in+cancer+in+association+with+promoter+hypermethylation.">Gene silencing in cancer in association with promoter hypermethylation.</a> New England Journal of Medicine. 349, 2042-2054 (2003).</li><li>Hanahan, D., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hallmarks+of+Cancer:+The+Next+Generation.">Hallmarks of Cancer: The Next Generation.</a> Cell. 144 (5), 646-674 (2011).</li><li>Howard, G., Eiges, R., Gaudet, F., Jaenisch, R., Eden, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+and+transposition+of+endogenous+retroviral+elements+in+hypomethylation+induced+tumors+in+mice.">Activation and transposition of endogenous retroviral elements in hypomethylation induced tumors in mice.</a> Oncogene. 27, 404-408 (2008).</li><li>Lander, E. 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P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relative+quantification+of+40+nucleic+acid+sequences+by+multiplex+ligation-dependent+probe+amplification.">Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification.</a> Nucleic Acids Research. 30, 57 (2013).</li><li>Nygren, A. O. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methylation-Specific+MLPA+(MS-MLPA):+simultaneous+detection+of+CpG+methylation+and+copy+number+changes+of+up+to+40+sequences.">Methylation-Specific MLPA (MS-MLPA): simultaneous detection of CpG methylation and copy number changes of up to 40 sequences.</a> Nucleic Acids Research. 33 (14), 128 (2005).</li><li>El Andaloussi, S., Mager, I., Breakefield, X. O., Wood, M. 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S., Yamamoto, Y., Sato, T., Ochiya, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+vesicles+mediate+the+horizontal+transfer+of+an+active+LINE-1+retrotransposon.">Extracellular vesicles mediate the horizontal transfer of an active LINE-1 retrotransposon.</a> Journal of Extracellular Vesicles. 8, 1643214 (2019).</li><li>Mannerstr&#246;m, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+alterations+in+mesenchymal+stem+cells+by+osteosarcoma-derived+extracellular+vesicles.">Epigenetic alterations in mesenchymal stem cells by osteosarcoma-derived extracellular vesicles.</a> Epigenetics. 14 (4), 352-364 (2019).</li><li>Shelke, G. V., L&#228;sser, C., Gho, Y. S., L&#246;tvall, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Importance+of+exosome+depletion+protocols+to+eliminate+functional+and+RNA-containing+extracellular+vesicles+from+fetal+bovine+serum.">Importance of exosome depletion protocols to eliminate functional and RNA-containing extracellular vesicles from fetal bovine serum.</a> Journal of Extracellular Vesicles. 3, 24783 (2014).</li><li>Wei, Z., Batagov, A. O., Carter, D. R., Krichevsky, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fetal+bovine+serum+RNA+interferes+with+the+cell+culture+derived+extracellular+RNA.">Fetal bovine serum RNA interferes with the cell culture derived extracellular RNA.</a> Scientific Reports. 6, 31175 (2016).</li><li>Kornilov, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+ultrafiltration-based+protocol+to+deplete+extracellular+vesicles+from+fetal+bovine+serum.">Efficient ultrafiltration-based protocol to deplete extracellular vesicles from fetal bovine serum.</a> Journal of Extracellular Vesicles. 7, 1422674 (2018).</li><li>Th&#233;ry, C., Amigorena, S., Raposo, G., Clayton, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+exosomes+from+cell+culture+supernatants+and+biological+fluids.">Isolation and characterization of exosomes from cell culture supernatants and biological fluids.</a> Current Protocols in Cell Biology. 30 (1), 1-29 (2006).</li><li>Puhka, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolomic+profiling+of+extracellular+vesicles+and+alternative+normalization+methods+reveal+enriched+metabolites+and+strategies+to+study+prostate+cancer-related+changes.">Metabolomic profiling of extracellular vesicles and alternative normalization methods reveal enriched metabolites and strategies to study prostate cancer-related changes.</a> Theranostics. 7 (16), 3824 (2017).</li><li>Peltoniemi, H. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+enrichment+does+not+warrant+a+higher+graft+survival+in+lipofilling+of+the+breast:+A+prospective+comparative+study.">Stem cell enrichment does not warrant a higher graft survival in lipofilling of the breast: A prospective comparative study.</a> Journal of Plastic, Reconstructive &amp; Aesthetic Surgery. 66 (11), 1494-1503 (2013).</li><li>Pavicic, W., Joensuu, E. I., Nieminen, T., Peltom&#228;ki, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LINE-1+hypomethylation+in+familial+and+sporadic+cancer.">LINE-1 hypomethylation in familial and sporadic cancer.</a> Journal of Molecular Medicine. 90, 827-835 (2012).</li><li>. L1.2 sequence on GenBank (accession number: AH005269.2) Available from: <a target="_blank" href="https://www.ncbi.nlm.nih.gov/nuccore/AH005269">https://www.ncbi.nlm.nih.gov/nuccore/AH005269</a> (2000)</li><li>Designing synthetic MLPA probes (v04). MRC-Holland Available from: <a target="_blank" href="https://support.mlpa.com/downloads/files/designing-synthetic-mlpa-probes">https://support.mlpa.com/downloads/files/designing-synthetic-mlpa-probes</a> (2018)</li><li>. Takara Bio Inc Available from: <a target="_blank" href="https://www.takarabio.com/us/products/cell_biology_and_epigenetics/epigenetics/dna_preparation/msre_overview">https://www.takarabio.com/us/products/cell_biology_and_epigenetics/epigenetics/dna_preparation/msre_overview</a> (2018)</li><li>Pisanic, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long+interspersed+nuclear+element+1+retrotransposons+become+deregulated+during+the+development+of+ovarian+cancer+precursor+lesions.">Long interspersed nuclear element 1 retrotransposons become deregulated during the development of ovarian cancer precursor lesions.</a> The American Journal of Pathology. 189 (3), 513-520 (2019).</li></ol>

This study illustrates how MSPA can be used to detect and quantify the methylation status of a specific genetic element. LINE-1 was the focus here, but the probes can be designed to target a range of genes and sequences. Moreover, there is a growing list of probe mixes available for different applications. MSPA is a simple and robust technique for DNA methylation analysis that does not require bisulfite conversion10. The complete procedure from sample preparation to data analysis takes around 2 da...

DNA methylation is a major epigenetic modification occurring in human cells. DNA methylation refers to the linkage of methyl groups to cytosine residues in CpG dinucleotides. Such dinucleotides are usually found in clusters (CpG islands) at the 5&#39; region of genes1. In normal cells, most of these dinucleotides exist in an unmethylated state, which allows DNA transcription. Incidentally, many cancers are associated with hypermethylated CpG islands and transcriptomic silencing2, especially in tumor suppressor genes, which in turn contribute to various hallmarks of cancer3.
On the other hand, long interspersed nuclear elements-1 (LINE-1s or L1s) are repetitive, transposable DNA elements that normally have high levels of methylation at CpG islands. Methylation of LINE-1 prevents translocation and helps maintain genome integrity. In several types of cancer, LINE-1 is hypomethylated, resulting in activation and subsequent retrotransposition-mediated chromosomal instability4. LINE-1 accounts for nearly 17% of the human genome5, and its methylation status may serve as an indicator of global genomic methylation levels6. Global LINE-1 hypomethylation is considered to precede the transition of cells to a tumor phenotype7; therefore, it holds promise as a potential marker for early cancer onset.
Currently, there are several methods for methylation analysis, including pyrosequencing, methylation-specific PCR, microarrays, and chromatin immunoprecipitation1. The use of next-generation sequencing has also made it possible to incorporate genome-wide approaches to detection of DNA methylation. Many of these methods rely on bisulfite-treated DNA, in which unmethylated cytosines are converted to uracil and methylated cytosines remain unchanged. However, working with bisulfite-treated DNA has several pitfalls, such as incomplete conversions of unmethylated cytosines to uracil, biased amplification of sequences, and sequencing errors8.
In methylation-specific probe amplification (MSPA), probes composed of two oligonucleotides target DNA sequences containing a restriction site (GCGC) for the methylation-sensitive restriction enzyme HhaI9. After the probes hybridize to DNA, each sample is divided into two sets. Probes in the first set undergo ligation, while probes in the second set undergo ligation followed by HhaI-mediated digestion at unmethylated CGCG sites. Both sets of samples are then amplified by PCR, and the products are separated by capillary electrophoresis. Probes at unmethylated sites are digested by HhaI and are not amplified during PCR, resulting in no peak signals. By contrast, probes at methylated sites are protected from digestion and are therefore amplified during PCR, subsequently generating peak signals10.
MSPA has several advantages over alternative methods. First, it requires a low amount of DNA (50&#8211;100 ng) and is well-suited for analysis of DNA from formalin-fixed paraffin embedded samples10. It does not require bisulfite-treated DNA; in fact, it is unsuitable for DNA that is modified in this way. Many samples can be analyzed at the same time, and MSPA probes can be designed such that they target multiple genes or sequences simultaneously. Additionally, the probes are specific and sensitive for methylated DNA as the HhaI restriction site corresponds to a sequence that is typical of CpG islands10.
This study investigated the effects of osteosarcoma (OS)-derived extracellular vesicles (EVs) on LINE-1 methylation in adipose tissue-derived mesenchymal stem cells (AT-MSCs; Figure 1). EVs are nanoscale, membrane-bound vesicles secreted by most cell types. They carry proteins, lipids, mRNA, microRNA, and additional molecules from parent cells11,12. EVs mediate intercellular communication and play important roles in several pathophysiological conditions13,14. A recent study showed that cancer-derived EVs may transfer active LINE-1 to recipient cells15. It has been reported earlier that EVs from the HOS-143B cell line can alter the methylation status of LINE-1 in MSCs, in addition to other genetic effects16.
When growing cells for EV isolation, it is important to use EV-depleted fetal bovine serum [FBS] in the growth medium, since FBS-derived EVs may interfere with EVs from other sources and hamper the results17,18. Ultracentrifugation is one of the most common methods for depleting EVs from FBS. It is a relatively simple and cost-effective procedure compared to alternatives such as ultrafiltration and commercial EV-depleted FBS19. Here, the protocol also demonstrates how to prepare EV-depleted FBS by ultracentrifugation.
This article presents a detailed protocol for the aforementioned techniques, from isolation of EVs from an OS cell line to the methylation analysis of LINE-1 in OS-EV treated MSCs (Figure 1).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 mL syringe</td>
			<td>Terumo</td>
			<td>SS+01T1</td>
			<td>for NTA</td>
		</tr>
		<tr>
			<td>24-well plate</td>
			<td>Corning</td>
			<td>3524</td>
			<td>MSC cell culture</td>
		</tr>
		<tr>
			<td>3730xl DNA Analyzer</td>
			<td>Applied Biosystems, ThermoFisher Scientific</td>
			<td>3730XL</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL centrifuge tube</td>
			<td>Corning</td>
			<td>430829</td>
			<td></td>
		</tr>
		<tr>
			<td>Beckman Optima LE-80K Ultracentrifuge</td>
			<td>Beckman</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BlueStar Prestained Protein Marker</td>
			<td>Nippon Genetics</td>
			<td>MWP03</td>
			<td>WB: protein marker</td>
		</tr>
		<tr>
			<td>Calnexin (clone C5C9)</td>
			<td>Cell Signaling Technology</td>
			<td>2679</td>
			<td>WB, dilution 1:800</td>
		</tr>
		<tr>
			<td>CD63 (clone H5C6)</td>
			<td>BD Biosciences</td>
			<td>556019</td>
			<td>WB, dilution 1:1000</td>
		</tr>
		<tr>
			<td>Centrifuge 5702 R</td>
			<td>Eppendorf</td>
			<td>5703000010</td>
			<td>For conditioned media and cells</td>
		</tr>
		<tr>
			<td>Centrifuge 5810</td>
			<td>Eppendorf</td>
			<td>5810000010</td>
			<td>For spinning down 96-well plate</td>
		</tr>
		<tr>
			<td>Centrifuge tube (polyallomer, 14x95 mm)</td>
			<td>Beckman</td>
			<td>331374</td>
			<td>Ultracentrifugation</td>
		</tr>
		<tr>
			<td>DMEM/F-12 + GlutaMAX medium</td>
			<td>Gibco, Life Technologies</td>
			<td>31331-028</td>
			<td>For AT-MSC culture</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco, Life Technologies</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr>
			<td>GeneScan 500 LIZ size standard</td>
			<td>Applied Biosystems, Life Technologies</td>
			<td>4322682</td>
			<td>for capillary electrophoresis</td>
		</tr>
		<tr>
			<td>GenomePlex Complete Whole Genome Amplification (WGA) Kit</td>
			<td>Sigma</td>
			<td>WGA2-10RXN</td>
			<td>for MSPA negative control</td>
		</tr>
		<tr>
			<td>Hi-Di formamide</td>
			<td>Applied Biosystems, Life Technologies</td>
			<td>4311320</td>
			<td>for capillary electrophoresis</td>
		</tr>
		<tr>
			<td>HOS-143B cell line</td>
			<td>ATCC</td>
			<td>CRL-8303</td>
			<td></td>
		</tr>
		<tr>
			<td>Hsp70 (clone 5G10)</td>
			<td>BD Biosciences</td>
			<td>554243</td>
			<td>WB, dilution 1:1000</td>
		</tr>
		<tr>
			<td>IRDye 800CW Goat anti-mouse</td>
			<td>Li-Cor</td>
			<td>926-32210</td>
			<td>WB: secondary</td>
		</tr>
		<tr>
			<td>IRDye 800CW Goat anti-rabbit</td>
			<td>Li-Cor</td>
			<td>926-32211</td>
			<td>WB: secondary</td>
		</tr>
		<tr>
			<td>LINE-1 probe-mix primers</td>
			<td>IDT</td>
			<td></td>
			<td>Sequences in Table 1</td>
		</tr>
		<tr>
			<td>MicroAmp Optical 96-well reaction plate with barcode</td>
			<td>Applied Biosystems, Life Technologies</td>
			<td>4306737</td>
			<td>also requires sealing film</td>
		</tr>
		<tr>
			<td>Micro BCA Protein Assay kit</td>
			<td>ThermoFisher Scientific</td>
			<td>23235</td>
			<td>measure protein concentration</td>
		</tr>
		<tr>
			<td>MiniProtean TGX 10% gels</td>
			<td>Bio-Rad</td>
			<td>456-1034</td>
			<td>WB: gel electrophoresis</td>
		</tr>
		<tr>
			<td>NanoSight LM14C</td>
			<td>Malvern Instruments</td>
			<td></td>
			<td>for NTA</td>
		</tr>
		<tr>
			<td>Nitrocellulose membrane 0.2 &#181;m</td>
			<td>Bio-Rad</td>
			<td>1620112</td>
			<td>WB: protein transfer</td>
		</tr>
		<tr>
			<td>NucleoSpin Tissue XS</td>
			<td>Macherey-Nagel</td>
			<td>740901.50</td>
			<td>for DNA extraction</td>
		</tr>
		<tr>
			<td>Odyssey Blocking Buffer</td>
			<td>Li-Cor</td>
			<td>927-40000</td>
			<td>WB: blocking, antibodies</td>
		</tr>
		<tr>
			<td>PBS, 1X</td>
			<td>Corning</td>
			<td>21-040-CVR</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Gibco, Life Technologies</td>
			<td>DE17-602E</td>
			<td>Antibiotics for culture media</td>
		</tr>
		<tr>
			<td>Protein LoBind tube, 0.5 mL</td>
			<td>Eppendorf</td>
			<td>22431064</td>
			<td>For storing Evs</td>
		</tr>
		<tr>
			<td>REVERT Total Protein Stain and Wash Solution Kit</td>
			<td>Li-Cor</td>
			<td>926-11015</td>
			<td>WB: total protein staining</td>
		</tr>
		<tr>
			<td>RKO cell line</td>
			<td>ATCC</td>
			<td>CRL-2577</td>
			<td>for MSPA positive control</td>
		</tr>
		<tr>
			<td>RPMI medium 1640 + GlutaMAX</td>
			<td>Gibco, Life Technologies</td>
			<td>61870-010</td>
			<td>For HOS-143B cell culture</td>
		</tr>
		<tr>
			<td>SALSA MLPA HhaI enzyme</td>
			<td>MRC-Holland</td>
			<td>SMR50</td>
			<td></td>
		</tr>
		<tr>
			<td>SALSA MLPA reagent kit</td>
			<td>MRC-Holland</td>
			<td>EK1-FAM</td>
			<td></td>
		</tr>
		<tr>
			<td>SALSA MLPA P300 probe-mix</td>
			<td>MRC-Holland</td>
			<td>P300-100R</td>
			<td></td>
		</tr>
		<tr>
			<td>Swinging rotor SW-28</td>
			<td>Beckman Coulter</td>
			<td>342207</td>
			<td>Ultracentrifugation</td>
		</tr>
		<tr>
			<td>Syringe filter, 0.22 &#181;m</td>
			<td>Jet Biofil</td>
			<td>FPE-204-030</td>
			<td>sterile filtering FBS</td>
		</tr>
		<tr>
			<td>Tecnai 12</td>
			<td>FEI Company</td>
			<td></td>
			<td>equipped with Gatan Orius SC 
			1000B CCD-camera 
			(Gatan Inc., USA); for TEM</td>
		</tr>
		<tr>
			<td>TBS, 1X tablets</td>
			<td>Medicago</td>
			<td>09-7500-100</td>
			<td>WB: buffer</td>
		</tr>
		<tr>
			<td>Trans-Blot Turbo</td>
			<td>Bio-Rad</td>
			<td></td>
			<td>WB: transfer</td>
		</tr>
		<tr>
			<td>Thermal cycler</td>
			<td>ThermoFisher Scientific</td>
			<td>TCA0096</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Express</td>
			<td>Gibco</td>
			<td>12604-021</td>
			<td>for trypsinization of cells</td>
		</tr>
		<tr>
			<td>TSG101 (clone 4A10)</td>
			<td>Sigma</td>
			<td>SAB2702167</td>
			<td>WB, dilution 1:500</td>
		</tr>
	</tbody>
</table>

line-1 methylation analysis in mesenchymal stem cells treated with osteosarcoma-derived extracellular vesicles

Methylation-specific probe amplification (MSPA) is a simple and robust technique that can be used to detect relative differences in methylation levels of DNA samples. It is resourceful, requires small amounts of DNA, and takes around 4&#8211;5 h of hands-on work. In the presented technique, DNA samples are first denatured then hybridized to probes that target DNA at either methylated or reference sites as a control. Hybridized DNA is separated into parallel reactions, one undergoing only ligation and the other undergoing ligation followed by HhaI-mediated digestion at unmethylated GCGC sequences. The resultant DNA fragments are amplified by PCR and separated by capillary electrophoresis. Methylated GCGC sites are not digested by HhaI and produce peak signals, while unmethylated GCGC sites are digested and no peak signals are generated. Comparing the control-normalized peaks of digested and undigested versions of each sample provides the methylation dosage ratio of a DNA sample. Here, MSPA is used to detect the effects of osteosarcoma-derived extracellular vesicles (EVs) on the methylation status of long interspersed nuclear element-1 (LINE-1) in mesenchymal stem cells. LINE-1s are repetitive DNA elements that typically undergo hypomethylation in cancer and, in this capacity, may serve as a biomarker. Ultracentrifugation is also used as a cost-effective method to separate extracellular vesicles from biological fluids (i.e., when preparing EV-depleted fetal bovine serum [FBS] and isolating EVs from osteosarcoma conditioned media [differential centrifugation]). For methylation analysis, custom LINE-1 probes are designed to target three methylation sites in the LINE-1 promoter sequence and seven control sites. This protocol demonstrates the use of MSPA for LINE-1 methylation analysis and describes the preparation of EV-depleted FBS by ultracentrifugation.

The main goal of this study was to evaluate the epigenetic effects of OS-EVs on MSCs. OS-EVs were isolated from HOS-143B cells using the standard differential centrifugation method. Expression of the typical EV markers CD63, Hsp70, and TSG101 by western blotting confirmed the presence of OS-EVs. (Figure 2A). Absence of calnexin signal indicated purity of the OS-EV isolate. Additional indication of purity was observed with TEM, with intact vesicles of various sizes being pres...

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LINE-1 Methylation Analysis in Mesenchymal Stem Cells Treated with Osteosarcoma-Derived Extracellular Vesicles

Described here is the use of a methylation-specific probe amplification method to analyze methylation levels of LINE-1 elements in mesenchymal stem cells treated with osteosarcoma-derived extracellular vesicles. Ultracentrifugation, a popular procedure for separating extracellular vesicles from fetal bovine serum, is also demonstrated.

Methylation-specific probe amplification (MSPA) is a simple and robust technique that can be used to detect relative differences in methylation levels of ...

DNA methylation is one of the most studied epigenetic signatures. Many cancers are characterized by changes in DNA methylation levels, which are associated with aberrant gene expression and other genomic abnormalities. Long interspersed nuclear elements are repetitive transposable genomic sequences that normally have high levels of methylation.However, they often become hypermethylated in cancer, which result in their activation and leads to chromosomal instability. In this study, we treated mesenchymal stem cells with osteosarcoma-derived extracellular vesicles to see whether cancer EVs can affect LINE-1 methylation in MECs. Fetal bovine serum, which is an integral component of mammalian cell culture media, contains a large number of bovine-derived EVs.These Evs will interfere with analysis of Evs from cells of our interest, therefore, when growing cells for EV isolation, it is important to use EV-depleted FBS. Take FBS in ultracentrifuge tubes and place them in ultracentrifuge buckets. Balance the buckets to within 10 milligrams of each other prior to loading them on a swinging rotor.Place the rotor in the ultracentrifuge and run at 100, 000 G for 19 hours at four degrees Celsius. After the run, carefully collect the light colored upper layer of the supernatant and transfer it to a 50 milliliter tube. Do not disturb or pipet the dark brown pellet as it contains EVs from FBS.Pass the supernatant through a 0.22 micron filter into a new 50 milliliter tube. This filter-sterilized, EV-depleted FBS can now be added to cell culture media when growing cells for EV isolation. Collect conditioned media from osteosarcoma cells grown in EV-depleted media.To remove cells and cell debris, centrifuge the conditioned media at 2500 G for 20 minutes at four degrees Celsius. Transfer the supernatant to ultracentrifuge tubes and balance them as earlier. Centrifuge the tubes at 100, 000 G for two hours at four degrees Celsius.Carefully discard the supernatant leaving around one milliliter at the bottom. Add around 20 milliliters of PBS to the tube and pipet gently in order to wash and re-suspend the EV pellet. Balance the tubes and carry out another round of ultracentrifugation with the same settings.Carefully remove the supernatant and re-suspend the EV pellet in 200 microliters of PBS by gentle pipetting. Store the EVs in low-binding tubes. Purified EVs are characterized by western blotting, nanoparticle tracking analysis, and transmission electron microscopy.Plate 15, 000 MSCs per well in a 24 well plate. After 24 hours, remove old media, wash the cells with PBS, and change to EV-depleted media. Treat cells with OS-EVs at the scheduled time points.After stopping the EV treatment, extract DNA from the cells using an appropriate method. For methylation analysis, we used a custom-made methylation-specific probe amplification method. Design the customized LINE-1 probes, as done previously, by Pavicic and others.For methylation probes, select three sequences containing the HhaI restriction site within the promoter region. For control probes, select seven sequences lacking the HhaI restriction site from the rest of the LINE-1 sequence. Dilute 70 nanograms of DNA sample in TE buffer to five microliters.Heat the samples for 10 minutes at 98 degrees Celsius then cool to 25 degrees Celsius. Add three microliters of probe hybridization mix to each sample and run the thermocycler to allow the probes to hybridize to DNA. At room temperature, add 30 microliters of post-hybridization mix to each sample.Transfer 10 microliters to a second tube. Place both sets of tubes in the thermocycler and incubate at 48 degrees Celsius for at least one minute. While the samples are at 48 degrees Celsius, add 10 microliters of ligation mix to the first set of tubes and 10 microliters of ligation-digestion mix to the second set of tubes.Run the next thermocylcer program. Then, spin down the tubes and simultaneously set the thermocycler to 72 degrees Celsius. Add five microliters of polymerase mix to each tube and place the tubes in the thermocycler.Run the PCR program. While the PCR program is running, prepare a solution of one milliliter formamide containing 2.5 microliters of size-standard. Pipet 10 microliters of this solution to each row of an optical 96 well plate.After PCR, dilute the undigested and the digested samples to one to one hundred and one to two hundred, respectively in ultra pure water. Add two microliters of diluted PCR product to the 96 well plate. Centrifuge the plate at 200 G for 15 to 20 seconds to remove air bubbles.Carry out fragment analysis of samples by capillary electrophoresis. Open the capillary electrophoresis results in an electropherogram analysis software. Select a sample and set the panel to MLPA from the menu.Click on the panel and press control D to apply this setting to all samples. In a similar way, set the analysis method to microsatellite default for all samples. Select all samples and click on the green play button to run analysis, then select all samples and click on the graph button to visualize the probe peaks.Zoom in on the peak region for a higher resolution of the individual peaks. Make sure that all 10 peaks corresponding to the probes from the LINE-1 probe mix are labeled. In the genotypes tab, export the results in the CSV format.Open the CSV file in a data analysis software and sort the data into columns. Label the three methylation site peaks based on their sizes. The remaining seven peaks correspond to the control probes.Here we are using the LINE-1 2M probe peaks which have a size of roughly 117 base pairs. For each sample, calculate the sum peak area of all seven control peaks. Divide the peak area of each LINE-1 probe by this sum.For each sample, divide the value of the digested sample by that of the undigested sample to obtain the methylation dosage ratio. Western blotting analysis confirmed the presence of OS-EVs with signals observed for TSG101, HSP70, and CD63. Absence of calnexin signal indicated that the EV sample was pure.Additional indication of purity was observed with TEM with intact vesicles of various sizes present in the OS-EV sample. The OS-EV particle concentration and size distribution was measured by NTA. 80%of the EVs were in the size range of 50 to 200 nanometers.Here we can see the methylation dosage ratios of LINE-1 from MSCs at different time points of the EV treatment. The gray line represents baseline methylation level from time point zero. At time point three, there is a decrease in the average methylation dosage ratio after administration of EVs, thereby indicating hypermethylation.This hypermethylating effect of the EVs is less pronounced at time point seven. Here we have shown a simple, highly sensitive, and robust technique for methylation analysis. It requires a low amount of DNA with no bisulfite conversion involved and it is also suitable for DNA isolated from paraffin-embedded samples.The whole method, with the experimental and analytical parts, takes around two days. Due to a high copy number of LINE-1 in the genome, the baseline methylation of LINE-1 is high in normal samples. Therefore, the differences in methylation levels of samples, as observed with this method, may be subtle.

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JoVE Journal

Genetics

5.6K vistas.  University of Helsinki. La metilación del ADN es una de las firmas epigenéticas más estudiadas. Muchos tipos de cáncer se caracterizan por cambios en los niveles de metilación del ADN, que se asocian con la expresión génica aberrante y otras anomalías genómicas. Los elementos nucleares intercalados largos son secuencias genómicas transponibles repetitivas que normalmente tienen altos niveles de metilación.Sin embargo, a menudo se hipermetilan en el cáncer, que resulta en su activación y conduce a...