DNA Methylation Analysis

2.7.1 Cell pellet preparation

The cell suspensions were generated as described in Section 2.2.3 and centrifuged at 3000 x g for 3 minutes with 1ml culture medium, supernatant removed, and the pellet washed twice with PBS by centrifugation, before the final supernatant removed and pellets stored at -20oC.

2.7.2 DNA extraction

DNA was extracted using the DNeasy Blood and Tissue kit (Qiagen) according to manufacturer’s instructions. Cell pellets were thawed and re-suspended in 200μl PBS, 20 μl proteinase K and 200μl buffer AL. Contaminant RNA was eliminated by RNase A treatment (Qiagen), followed by vortexing and incubated at 56˚C in water bath for approximately 10 minutes for lysing. After incubation, 200μl of 96% ethanol was added, mixed well, and pipetted into a DNeasy spin column in a 2ml collection tube and centrifuged at 8000 rpm for 1 minute. The flow-through was removed leaving total DNA attached to the DNeasy column membrane and then the spin column transferred into a new 2ml collection tube, washed twice, with 500µl buffer AW1 and then with 500µl Buffer AW2, by centrifugation at 8000 rpm and 13000 rpm for 1 and 3 minutes, respectively. Flow-through was discarded and the spin column placed into a new 2ml microcentrifuge tube and DNA eluted in 50μl DNase and RNase free water by centrifugation at 8000 rpm for 1 minute. This step was repeated twice

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to increase the yield of extracted DNA. The integrity and concentration of DNA was determined by running 200ng DNA on 1% agarose gels using ethidium bromide staining with 1x electrophoresis buffer TAE at 100 V for 1 hour (Figure 2.3) and by spectrophotometrically analysis at 260 nm with a NanoDrop 2000/2000c (Thermo Scientific). Extracted DNA was stored at -20°C.

Figure 2.3 Example agarose gel electrophoresis. The quality and integrity of genomic

DNA (approximately 200 ng DNA) extracted from hESCs samples incubated in normoxic (21%ST) and hypoxic (2%PG and 2%WKS) oxygen conditions using standard gel electrophoresis. A: Undifferentiated cells; B: Differentiated cells (5 Days); C: Differentiated cells (10 Days); D: Differentiated cells (20 Days); M: DNA marker. Gel images were captured with Syngene Genesnap software.

2.7.3 DNAs global methylation

2.7.3.1 Elisa-based methods

Quantification of total 5mC content within DNA isolated from cell lines was performed using the MethylFlashTM methylated DNA quantification Kit (Epigentek). In brief, the DNA sample is captured in a pre-coated 96-well ELISA plate, and the methylation status detected after several incubation steps with primary and secondary antibody before quantification of

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methylated DNA by reading calorimetrically at A450 and the quantification of global DNA methylation derived from the absorbance measured(Kurdyukov and Bullock, 2016).

2.7.3.2 5-Methylcytosine (5mC)

The ELISA analysis was performed following manufacturer’s instructions; 100ng of genomic DNA for each sample (in triplicate) was used in the assay. First, a standard curve of methylated polynucleotide containing 50% of 5-methylcytosine as a positive control was generated using the five concentrations indicated into Table 2.2, and these were added to their respective wells.

Table 2.2 Summary of the five-different concentration of the positive control used to generate the standard curve.

Standards Concentration of Positive control

1 0.5ng/μl

2 1.0ng/μl

3 2.0ng/μl

4 5.0ng/μl

5 10.0ng/μl

100ng of genomic DNA for each sample was quantified as previously described in Section

2.7.2 and diluted to a final volume of 10μl with distilled water. Binding solution (80μl) was

added to each well of 48 well plates. 1μl of negative control buffer (an unmethylated polynucleotide), standard and the sample DNA was added to the respective wells and mixed well by pipetted several times to ensure the solution coated the bottom of the well (Figure

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Figure 2.4 A schematic of the 48 well plate. The negative control and standards were

loaded in duplicates. The samples were loaded in the remaining wells.

Then, the binding solution was removed, and the plate washed three times with 150μl of 1X

wash buffer (prepared by diluted 10X wash buffer (ME1) diluted with 117ml of distilled

water). Further, 50µl of a 1:1000 dilution of capture antibody was added to each well and incubated for 60 minutes at room temperature. After incubation the solution was removed before a further three washes with 150μl of wash buffer was performed again. Then 50µl of detection antibody (1:2000) was added to each well followed by incubation at room temperature for 30 minutes prior to the solution being removed and a further four washes with 150μl wash buffer. Next 50μl of enhancer buffer (diluted at a ratio of 1:5000) was added and samples incubated at room temperature for 30 minutes before removal and washed for five times, as above. Following washing, 100μl of developer solution (1:5000) was added and plates incubated at room temperature for 10 minutes in the dark. The developer solution acted as a chromogenic reagent which turned blue in the presence of sufficient methylated DNA. Following this 50μl of stop solution was added to stop the colorimetric reaction and the plate placed on a shaking frame to ensure that the reaction was completely stopped.

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Absorbance was measured at 450 nm with a micro-plate reader (BioTek, Synergy 2) using the program Gen5 1.10.

Standard curve was determined using linear regression (Figure 2.5) and the percentage of 5mC in the total DNA calculated using the following formula:

Where 2* are a normalization factors in the positive control to 100%, as the positive control contains only 50% of 5mC. S is the amount of input sample DNA in ng.

2.7.3.3 5-Hydroxymethylcytosine (5hmC)

Similar to the Methylated DNA Quantification kit protocol, 200ng amount of isolated DNA was quantified using the colorimetric MethylFlashTM Hydroxymethylated DNA Quantification Kit (Epigentek). Linear regression was performed on the standard curve generated from the positive controls and the total amount (Figure 2.5) and percentage of methylated (5hmC) DNA quantified using the following formula:

5* is a factor to normalize 5hmC in the positive control to 100%, as the positive control contains only 20% of 5hmC. S is the amount of input sample DNA in ng.

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Figure 2.5 Example standard curve of determination of DNA methylation by the immunoassay. A) Linear relationship between the absorbance and amount of 5-

methylcytosine. B) Linear relationship between the absorbance and amount of 5- hydroxymethylcytosine.