Methylation analyses

DNA methylation analyses were performed using the bisulphite modification protocol developed by Frommer and colleagues (Frommer et al., 1992). This method involves bisulphite modification, which is the conversion of unmethylated, but not methylated (5-methylcytosine), cytosine residues to uracil by deamination. This provides a robust method which allows for the distinction between methylated and unmethylated DNA. Sequencing primers for a region of interest, commonly CpG islands known to contain several CpG sites, are designed and used to amplify the region of interest by PCR of the bisulphite- modified (BM) DNA.

Following amplification of the BM DNA, DNA methylation was quantified at specific CpG sites by pyrosequencing, a ‘sequencing by synthesis’ technique regarded as the gold standard (Migheli et al., 2013). Pyrosequencing involves the quantification of methylation at individual cytosine residues within a specific region of interest, expressed as percentage methylation. Methylation analyses were performed using commercially-available, optimised Qiagen assays, which include both the PCR primer and sequencing primer. Details about the sequences analysed and primers can be found in sections 3.5 and 5.4.3).

Firstly, the denatured single-stranded PCR product sequence is hybridised by the sequencing primer and is elongated by DNA polymerase with the addition and incorporation of nucleotides. When a deoxynucleotide triphosphate (dNTP) is added, a molecule of pyrophosphate (PPi) is released (Figure 2.6 (A)). This pyrophosphate is then converted into adenosine triphosphate (ATP) by sulfurylase. Luciferin is oxidised by luciferase using the generated ATP (Figure 2.6 (B)), and this produces a light signal that is detected by the pyrosequencer and expressed as a peak for the incorporated nucleotide on the Pyrogram® (Figure 2.6 (C)). If the nucleotide was not incorporated, it is degraded by apyrase and there is no subsequent generation of ATP or resultant peak. The heights of the resultant peaks for each dNTP on the Pyrogram are proportional to the number of nucleotides added.

Figure 2.6 Steps involved in the quantification of DNA methylation by pyrosequencing.

Adapted with permission from Qiagen (Qiagen, 2014).

2.2.3.1 DNA extraction

DNA was extracted from half a rectal mucosal biopsy using a phenol-chloroform protocol. OCT-embedded samples were removed from storage at -80°C and excess OCT compound was removed. Whole biopsies were cut in half using a new, sterile scalpel blade for each sample and the remaining half not used was immediately placed back into its tube and returned to storage at -80°C.

Samples were homogenised on a shaker set to 900rpm for 8 hours at 55°C in 500μl of SET-sodium dodecyl sulphate solution (25ml Tris (50mM), 12.5Mm EDTA, 0.5% sodium dodecyl sulphate (SDS)) with the addition of 16μl of Proteinase K (Fermentas). The homogenised samples were centrifuged at 13,000rpm for 3 minutes. DNA was precipitated using 600μl of chloroform:isoamyl alcohol 24:1 (Fermentas). Phase lock gel (5 Prime, Hamburg, Germany) was utilised to separate the DNA by centrifuging at 10,000rpm for 5 minutes at room temperature. The upper phase was poured into a new tube and 16μl of RNase A/T1 (Fermentas) were added. After incubating at 37°C for 30 minutes, 42μl of sodium acetate solution (3M, pH 5.2) were added. The tubes were inverted several times and 400μl of isopropanol

and 2.5μl of glycogen (Fermentas) were added. The tubes were inverted several times and then centrifuged at 13,000rpm for 5 minutes at room temperature. The supernatant was discarded and the DNA pellet was washed twice by adding 500μl of 70% ethanol and centrifuging at 13,000rpm for 2 minutes at room temperature, discarding the ethanol in-between washes. The DNA pellet was left to air dry for an hour, re-suspended in 50μl of 2mM Tris and left to dissolve overnight at 4°C on the shaker.

The DNA purity and concentration were measured using the NanoDrop 1000 spectrophotometer, similarly to that described for RNA in Section 2.2.1.1.1. DNA extraction from the Intervention, ‘UC’ and ‘Polyp’ biopsy samples was performed by Mr. Iain McCallum, Dr. Naomi Willis and Dr. Long Xie.

2.2.3.2 Bisulphite modification of DNA

Bisulphite modification of DNA was performed using the EZ DNA Methylation- Gold™ Kit (Zymo Research). A total of 250ng of DNA made up to a volume of 20μl with water was prepared and 130μl of CT Conversion Reagent (900μl water, 300μl M-Dilution Buffer and 50μl M-Dissolving Buffer) were added. The samples were mixed by pipetting up and down, centrifuged and placed in the Sensoquest lab cycler programmed to perform the following steps for DNA denaturation followed by CT-conversion reaction:

1. 98°C for 10 minutes 2. 64°C for 2.5 hours 3. Hold at 4°C for storage

DNA samples were loaded into Zymo-Spin™ IC Columns containing 600μl of M-Binding Buffer placed in a collection tube. These were centrifuged at maximum speed and the flow-through discarded. Samples were washed by adding 100μl of M-Wash Buffer and centrifuging at maximum speed for 30 seconds. The samples were then incubated at room temperature with 200μl of M-Desulphonation Buffer for 20 minutes. After the incubation, samples were centrifuged at maximum speed for 30 seconds. Two further wash steps were performed by adding 200μl of M-Wash Buffer, centrifuging at maximum speed for 30 seconds and repeating. The columns were placed into 1.5ml

microcentrifuge tubes, and to elute the BM DNA 10μl of M-Elution Buffer were added followed by a centrifugation step for 30 seconds at maximum speed. DNA bisulphite modification reactions of the Intervention samples were performed Dr. Naomi Willis and Dr. Long Xie as part of the DISC Study.

2.2.3.3 Amplification of bisulphite-modified DNA

A PCR master mix was prepared as described in Table 2.19 and 24μl were dispensed into 0.5ml tubes. BM DNA (1μl) was added to each tube, with each sample performed in duplicate, with the exception of the negative controls which included 1μl of Epitect human control DNA (Qiagen) or 1μl of water. All PCR reactions were performed on the S1000™ (Bio-Rad) PCR machine under the conditions described in Table 2.20.

Table 2.19 Pyrosequencing PCR reaction components.

Component Volume per reaction (µl)

HotStarTaq Master Mix (2x) 12.5

H2O 9.5

PCR primer 2

Table 2.20 Cycling conditions for pyrosequencing PCR.

Step Time Temperature Number of cycles

Initialization 15 minutes 95°C 1 3-step cycling: Denaturation Annealing Extension 20 seconds 40 seconds 20 seconds 95°C 55°C 72°C 40

2.2.3.3.1 Agarose gel electrophoresis of PCR products for methylation analyses

PCR products and negative controls were run on a 1% agarose gel stained with SafeView (NBS Biologicals Ltd, Cambridgeshire, UK) by loading 4μl of PCR products with 1μl of loading dye alongside 3μl of 100bp DNA ladder. Samples were electrophoresed at 120mV, 300mA for 35 minutes to check that the correct sequence was amplified and that there was no contamination in the negative controls (H2O and gDNA) (Figure 2.7).

Figure 2.7 Agarose gel electrophoresis image of SFRP1 assay PCR product.

Agarose gel electrophoresis was used to check the PCR products prior to analysis by pyrosequencing. There were no bands present in the negative controls (H2O and

gDNA) indicating that there was no contamination. Bands of the correct product size (257bp) were observed for all sample and control PCR products.

2.2.3.4 Pyrosequencing

Pyrosequencing was performed using the Pyromark Q96 ID (Qiagen) Pyrosequencer and Pyromark Gold Q96 reagents (Qiagen). A master mix for the 96-well PCR plate was prepared as described in Table 2.21, and 70μl of master mix were added to each well followed by 5μl of PCR product. The PCR plate was sealed and shaken for 10 minutes at 15rpm to prevent sedimentation of the beads.

Table 2.21 Pyrosequencing master mix for PCR plate.

Component Volume per reaction

(µl)

Binding Buffer (Qiagen) 38

Nuclease-free water (Qiagen) 30

Streptavidin Sepharose High Performance

Beads (GE Healthcare, Uppsala, Sweden) 2

In a pyrosequencing plate (Qiagen), 9μl of annealing buffer (Qiagen) and 3μl of sequencing primer (Qiagen) were added to each well. The PyroMark Q96 Vacuum Workstation (Qiagen) was used to aspirate the samples from the PCR plate using the vacuum. The vacuum probes were then washed in 70% ethanol for 5 seconds, denatured in denaturing buffer (0.8% NaOH solution) for 5 seconds and washed in Pyromark wash buffer (Qiagen) for 5 seconds. The vacuum was switched off and the samples were transferred to the pyrosequencing plate and heated at 80°C for at least 2 minutes to bind the sequencing primer. The enzyme, substrate mixtures and nucleotides were dispensed into their respective reagent and nucleotide tips as determined by the PyroMark CpG Software 1.0.11. The cartridge and plate were inserted and, after successful tip dispensation testing, run on the pyrosequencer.

All samples were run in duplicate using duplicates from two individual PCR amplification reactions. Participant sample order was randomised so that comparable numbers of each gender and endoscopy procedure, and intervention group as appropriate, were present on each pyrosequencing run plate. Pre- and post-intervention samples were run on the same pyrosequencing plate for the intervention samples. On each pyrosequencing plate, two negative controls to check for any contamination and 0% and 100% methylation controls were run alongside the samples.

2.2.3.5 Bisulphite modification quality control

Quantification of methylation by pyrosequencing included an internal control that confirmed full bisulphite conversion i.e. that all templates show conversion to thymine where unmethylated cytosines are not followed by guanine. Any samples indicating an incomplete bisulphite conversion were flagged by the Pyromark software (highlighted in yellow) at the analysis stage and would be consequently excluded from subsequent analyses. I did not encounter this problem during my analyses.

As, in a minority of cases, previously bisulphite-modified and stored DNA samples were utilised for quantification of methylation by pyrosequencing, methylation was quantified and compared in newly bisulphite-modified and previously bisulphite-modified samples. The results from this analysis showed there were no differences in the amplification of BM DNA by PCR and that methylation levels of SFRP1 at each CpG site were highly comparable between both newly- and previously- bisulphite-modified samples (see Table 2.22). Figure 2.8 Agarose gel electrophoresis image for comparison of SFRP1 assay PCR products amplified from 091 pre-intervention newly bisulphite- modified samples and previously bisulphite-modified samples.

Agarose gel electrophoresis was used to check the PCR products prior to analysis by pyrosequencing. In this image, PCR products from newly bisulphite-modified (N) and previously bisulphite-modified (P) DNA from sample 091 pre-intervention are compared. Bands of the correct product size (257bp) were observed for both samples and were of comparable intensity.

Table 2.22 Comparison of 091 pre-intervention SFRP1 methylation levels using newly bisulphite-modified samples and previously bisulphite- modified samples.

SFRP1 methylation (%)

Mean (SD)

CpG site 091 pre N 091 pre P

1 27.3 (3.2) 26.2 (0.6) 2 21.6 (1.8) 21.5 (2.3) 3 22.0 (2.9) 18.9 (0.6) 4 29.2 (0.2) 24.8 (4.3) 5 19.4 (1.3) 19.4 (1.4) 6 26.1 (3.2) 24.5 (2.4) 7 25.6 (2.4) 24.9 (1.7) Mean 24.4 (3.5) 22.9 (2.9)

SFRP1 methylation levels (%) at each CpG site and the mean methylation across all

seven CpG sites expressed as mean and standard deviation for newly bisulphite- modified (N) and previously bisulphite-modified (P) samples for 091 pre-intervention.

2.2.3.6 Methylation data processing

The means of the duplicates of the pyrosequencing data, expressed as percentage methylation (%), were taken and used for statistical analyses. Duplicate data were checked for a difference of no greater than 5%. Samples with a discordance between duplicates of >5% were repeated, and subsequently excluded from analyses if a difference of ≤5% was not achieved.