2.4 Discussion
3.2.2 Agilent custom-designed 8x15k microarray
Target selection
The workflow used to select the targets on the microarrays is shown in Figure 3.1. The loci targeted by the oligos on the microarray were mainly selected from our previous work and the published literature associated with acquired drug resistance: 1) CGIs identified from the DNA methylation profiling study by the DMH assay on a HCGI12K microarray between A2780 sensitive and resistant derivatives generated in vitro as well as between in
vivogenerated cell line pairs (PEO1 vs. PEO4, PEA1 vs. PEA2, PEO14 vs. PEO23) per-
formed by Dr. Jens M. Teodoridis; 2)a prognostic DNA methylation signature associated with progression free survival in ovarian cancer (Wei et al., 2006); 3) and 4) CGIs identified from DNA methylation profiling study on an Orion MethylScope (Lippman et al., 2004) two-channel microarray based on McrBC restriction enzyme to detect differential methy- lation between A2780 sensitive and resistant cell lines (data not published); 5) candidate genes collected from the studies about acquired drug resistance in ovarian cancer through our collaborations (Professor Hani Gabra and Dr. Euan A. Stronach from Ovarian Cancer Action); and 6) other loci/genes differentially expressed before and after cisplatin treatment from gene expression profiling studies published from 2001 to 2008 (see Appendix 3.1 for the gene list and reference). Promoter CGIs within 2kb of the transcription start site (TSS) of the selected genes were defined by Gardiner-Garden (1987) by the following criteria: CG content greater than 50%, length of sequence over 200bp and the ratio of observed to expected CpGs is larger than 0.6. The genes without any promoter CGI were not taken into consideration. The CGIs that were not close to the TSS of the genes were also selected in the study if they showed differential methylation between the in vitro and in vivo generated cisplatin sensitive and resistant cell lines identified by MLDA on HCGI12K arrays (see Chapter 2) or on Orion MethylScope microarrays (data not published). In total, about 600
3.2 Methods 96 loci were covered by the custom-designed microarray.
Controls on the microarray
Three major types of controls were designed on the microarray (Table 3.1). Firstly, the sequences from the human mitochondrial genome which are generally unmethylated in human (see Chapter 2), and sequences from chromosome 16 that are underrepresented for CG regions and which contain very few McrBC recognition sites (ACG or GCG) were selected as unmethylated controls. Secondly, the sequences on HCGI12K microarray (see Chapter 2) that showed high methylation levels in our previous DMH work were selected as positive methylation controls. Finally, sequences from non-human genome (see Table 3.1) were used as the negative controls which mainly reflected the background noise.
Controls # probes Source
Unmethylation 266 Mitochondrial genome
Unmethylation 400 Chr16: 59000000-61000000
Methylation 172 methylated probes on UHN12K microarray;
promoter CGIs of GAGE family
Negative 577 Structural negatives (3xSLv1)
1
Biological negatives2
Table 3.1: Controls on the custom-designed microarray.1This probe forms a hairpin and
does not hybridize well with labelled samples of any species.-2This probe has been shown
Chapter 3. Feasibility Study of DNA Methylation Profiling on Agilent
Custom-designed Microarray in Ovarian Cancer Cell Lines 97
Figure 3.1: Workflow of target selection on Agilent custom-designed microarray in the fea-
sibility study. 1CGIs identified from DNA methylation profiling study by DMH assay on
HCGI12K microarray between A2780 sensitive and resistant derivatives generated in vitro as well as between in vivo generated cell line pairs (PEO1 vs. PEO4, PEA1 vs. PEA2,
PEO14 vs. PEO23) performed by Dr. Jens M. Teodoridis; 2the prognostic DNA methy-
lation signature associated with progression free survival in ovarian cancer (Wei et al.,
2006);3,4CGIs identified from DNA methylation profiling study on an Orion MethylScope
(Lippman et al., 2004) two-channel microarray based on McrBC restriction enzyme to detect differential methylation between A2780 sensitive and resistant cell lines (data not
published);5candidate genes collected from the studies about acquired drug resistance in
ovarian cancer through our collaborations (Professor Hani Gabra and Dr. Euan A. Stonach
from Ovarian Cancer Action); 6other loci/genes differentially expressed before and after
3.2 Methods 98
Annotation of custom-designed microarray
The genomic annotation of human genes was obtained from the Refseq database (http: //www.ncbi.nlm.nih.gov/RefSeq/) and the UCSC known gene database (http: //genome.ucsc.edu/). Promoter CGIs within a 2kb of the transcription start site of the genes were obtained from UCSC database (Rhead et al., 2010) and from a genome- wide prediction of CGIs (Bock et al., 2007) as a supplementary set, both of which fulfil the mathematical model proposed by Gardiner-Garden to define a CGI (Gardiner-Garden and Frommer, 1987). MseI fragments were predicted by searching for the recognition sites (T/TAA) against the whole human genome sequence, masking the assembly gaps, intro- contig ambiguities and repeat regions. The prediction was performed in R using two pack- ages: BSgenome and BSgenome.Hsapiens.UCSC.hg18. The genomic positions of targets were specified by Human Mar. 2006 (NCBI36/hg18) assembly. All the information was stored in an in-house MySQL database (http://www.mysql.com/). An R package, RODBC, provides an interface between R workspace and MySQL database.
The annotation pipeline is shown in Figure 3.2. The genomic locations of the probes were compared to the transcript start sites of the genes collected from Refseq database and UCSC known gene database, the genomic location of CGIs and MseI fragments. Any gene for which the TSS was within 2kb span of the probes was added into the annotation table. If multiple transcripts or different genes were related to the same probe, the annotation table included multiple official gene symbols, Entrez gene IDs, TSSs, and strands in the record for the corresponding probe. The genomic location and length of CGIs and MseI fragments targeted by the probes were also added in the annotation table. If the probes were in the 150nt flanking region of a CGI, it was annotated as a ”flanking” probe. R functions [mseI.anno( ), cgi.anno( ) and gene.anno( )] for the microarray annotation are shown in the
Chapter 3. Feasibility Study of DNA Methylation Profiling on Agilent
Custom-designed Microarray in Ovarian Cancer Cell Lines 99
Figure 3.2: Annotation workflow of Agilent custom-designed microarray. *The probes are annotated as NA (not available) if they cover the MseI recognition site (T/TAA). CGI database 1 is from UCSC database and CGI database 2 is from Bock et al. (2007)
Appendix scripts and a complete annotation of this array is given in Appendix 3.2.