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