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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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.

Protocol

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

  1. 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.
  2. Load the buckets on a swinging rotor (type SW28, k-factor 246). Place the rotor in the ultra.......

Representative Results

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.......

Discussion

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.......

Acknowledgements

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.

....

Materials

NameCompanyCatalog NumberComments
1 mL syringeTerumoSS+01T1for NTA
24-well plateCorning3524MSC cell culture
3730xl DNA AnalyzerApplied Biosystems, ThermoFisher Scientific3730XL
50 mL centrifuge tubeCorning430829
Beckman Optima LE-80K UltracentrifugeBeckman
BlueStar Prestained Protein MarkerNippon GeneticsMWP03WB: protein marker
Calnexin (clone C5C9)Cell Signaling Technology2679WB, dilution 1:800
CD63 (clone H5C6)BD Biosciences556019WB, dilution 1:1000
Centrifuge 5702 REppendorf5703000010For conditioned media and cells
Centrifuge 5810Eppendorf5810000010For spinning down 96-well plate
Centrifuge tube (polyallomer, 14x95 mm)Beckman331374Ultracentrifugation
DMEM/F-12 + GlutaMAX mediumGibco, Life Technologies31331-028For AT-MSC culture
Fetal bovine serumGibco, Life Technologies10270-106
GeneScan 500 LIZ size standardApplied Biosystems, Life Technologies4322682for capillary electrophoresis
GenomePlex Complete Whole Genome Amplification (WGA) KitSigmaWGA2-10RXNfor MSPA negative control
Hi-Di formamideApplied Biosystems, Life Technologies4311320for capillary electrophoresis
HOS-143B cell lineATCCCRL-8303
Hsp70 (clone 5G10)BD Biosciences554243WB, dilution 1:1000
IRDye 800CW Goat anti-mouseLi-Cor926-32210WB: secondary
IRDye 800CW Goat anti-rabbitLi-Cor926-32211WB: secondary
LINE-1 probe-mix primersIDTSequences in Table 1
MicroAmp Optical 96-well reaction plate with barcodeApplied Biosystems, Life Technologies4306737also requires sealing film
Micro BCA Protein Assay kitThermoFisher Scientific23235measure protein concentration
MiniProtean TGX 10% gelsBio-Rad456-1034WB: gel electrophoresis
NanoSight LM14CMalvern Instrumentsfor NTA
Nitrocellulose membrane 0.2 µmBio-Rad1620112WB: protein transfer
NucleoSpin Tissue XSMacherey-Nagel740901.50for DNA extraction
Odyssey Blocking BufferLi-Cor927-40000WB: blocking, antibodies
PBS, 1XCorning21-040-CVR
Penicillin-streptomycinGibco, Life TechnologiesDE17-602EAntibiotics for culture media
Protein LoBind tube, 0.5 mLEppendorf22431064For storing Evs
REVERT Total Protein Stain and Wash Solution KitLi-Cor926-11015WB: total protein staining
RKO cell lineATCCCRL-2577for MSPA positive control
RPMI medium 1640 + GlutaMAXGibco, Life Technologies61870-010For HOS-143B cell culture
SALSA MLPA HhaI enzymeMRC-HollandSMR50
SALSA MLPA reagent kitMRC-HollandEK1-FAM
SALSA MLPA P300 probe-mixMRC-HollandP300-100R
Swinging rotor SW-28Beckman Coulter342207Ultracentrifugation
Syringe filter, 0.22 µmJet BiofilFPE-204-030sterile filtering FBS
Tecnai 12FEI Companyequipped with Gatan Orius SC
1000B CCD-camera
(Gatan Inc., USA); for TEM
TBS, 1X tabletsMedicago09-7500-100WB: buffer
Trans-Blot TurboBio-RadWB: transfer
Thermal cyclerThermoFisher ScientificTCA0096
TrypLE ExpressGibco12604-021for trypsinization of cells
TSG101 (clone 4A10)SigmaSAB2702167WB, dilution 1:500

References

Explore More Articles

LINE 1 MethylationMesenchymal Stem CellsOsteosarcomaExtracellular VesiclesDNA MethylationEpigeneticsUltracentrifugationEV IsolationFBS Depletion

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