A Method to Evaluate Genome-Wide Methylation in Archival Formalin-Fixed, Paraffin-Embedded Ovarian Epithelial Cells

Paraffin-Embedded Ovarian Epithelial Cells. PLoS ONE 9(8): e104481. doi:10.1371/journal.pone.0104481 A Method to Evaluate Genome-Wide Methylation in Archival Formalin-Fixed, Paraffin-Embedded Ovarian Epithelial Cells Qiling Li 0 Min Li 0 Li Ma 0 Wenzhi Li 0 Xuehong Wu 0 Jendai Richards 0 Guoxing Fu 0 Wei Xu 0 Tameka Bythwood 0 Xu Li 0 Jianxin Wang 0 Qing Song 0 Sudha Agarwal, Ohio State University, United States of America 0 1 Department of Obstetrics and Gynecology, First Affiliated Hospital, Xi'an Jiaotong University , Xi'an, Shaanxi , China , 2 Cardiovascular Research Institute, Morehouse School of Medicine, Atlanta, Georgia, United States of America, 3 School of Information Science and Engineering, Central South University , Changsha , China Background: The use of DNA from archival formalin and paraffin embedded (FFPE) tissue for genetic and epigenetic analyses may be problematic, since the DNA is often degraded and only limited amounts may be available. Thus, it is currently not known whether genome-wide methylation can be reliably assessed in DNA from archival FFPE tissue. Methodology/Principal Findings: Ovarian tissues, which were obtained and formalin-fixed and paraffin-embedded in either 1999 or 2011, were sectioned and stained with hematoxylin-eosin (H&E).Epithelial cells were captured by laser micro dissection, and their DNA subjected to whole genomic bisulfite conversion, whole genomic polymerase chain reaction (PCR) amplification, and purification. Sequencing and software analyses were performed to identify the extent of genomic methylation. We observed that 31.7% of sequence reads from the DNA in the 1999 archival FFPE tissue, and 70.6% of the reads from the 2011 sample, could be matched with the genome. Methylation rates of CpG on the Watson and Crick strands were 32.2% and 45.5%, respectively, in the 1999 sample, and 65.1% and 42.7% in the 2011 sample. Conclusions/Significance: We have developed an efficient method that allows DNA methylation to be assessed in archival FFPE tissue samples. - Funding: This work was supported by National Institutes of Health grants [HL003676, RR014758, RR003034]; American Heart Association grant [09GRNT2300003]; and in part by the Baltimore Research Enhancement Award Program in Stroke and the Baltimore Geriatrics Research, Education, and Clinical Center of the Department of Veterans Affairs. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Epigenetics is the study of heritable changes in gene expression that are not attributable to alterations in the DNA sequence. DNA methylation is a well-known epigenetic marker that plays an important role in the control of gene activity and the architecture of the nucleus [1]. Evaluation of the level of methylation at all cytosine nucleotides in an individuals genome (called the methylome) has recently become possible with the advent of next generation sequencing techniques, specifically sodium bisulfite sequencing [2,3]. Many different PCR-based methods for the detection of DNA methylation have been developed [4]. Archived human tissues, with known clinical follow-up, represent a valuable resource, particularly for retrospective genetic and epigenetic studies and identification of biological markers that might be useful for risk prediction of disease or prognosis [5]. Biopsied or surgically excised tissues obtained for routine histopathological analysis and diagnosis are widely available, and often formalin and paraffin embedded (FFPE) for decades. However, to date there is no suitable platform to assay genomewide methylation in these widely available samples. With the increasing interest in understanding the genetic and epigenetic basis of diseases, the ability to extract DNA from these FFPE samples represents an invaluable source of diagnostic material that can be used for genomic analyses and translational studies. Using DNA from old FFPE tissue may be problematic, as the DNA is often degraded and only limited amounts may be available. The quality of FFPE specimens decreases with time [6] because of linking of nucleic acids and proteins, as well as fragmentation of nucleic acids [5]. Historically, FFPE samples were not considered as a viable source for molecular analyses because the nucleic acids may be heavily modified by proteinnucleic acid and protein-protein cross linking [4]. Furthermore, sodium bisulfite treatment [6], which preserves methylation marks, and as such is necessary for PCR-based studies of DNA methylation, may further degrade the DNA. However, the effect of storage time on the ability to detect genome-wide methylation in FFPE tissues has not yet been documented. Laser-assisted microdissection is a proven method for isolating specific cell populations for molecular profiling [7]. It can cut particular cells of interest from a tissue section attached to an underlying membrane. In the present study, the identification of ovarian epithelial cells relied on morphological cell characteristics observed after routine histological staining. The system is based on an infrared laser that captures the ovarian epithelial cells of interest from tissue sections mounted on glass slides [7]. In this study, we extracted DNA from epithelial cells within human ovarian FFPE tissues that were obtained from two different individuals and stored for different numbers of years. Using our DNA extraction method, we tested and compared the whole genomic DNA methylation levels in these differentially aged samples using a series of steps including bisulfite conversion, whole genome amplification, purification of amplification, sequencing using a Junior 454 sequencer, and bioinformatics analysis. Overview of ovarian epithelial cell methylation To compare the genome-wide DNA methylation patterns in FFPE ovarian tissue samples stored since 1999 (O1999) or 2011 (O2011), we analyzed their methylation status after bisulfite treatment. The optimized workflow for DNA methylation analysis is summarized in Figure 1, and described in more detail in the Methods. Ten-micron tissue sections were prepared and stained with H&E. Epithelial cells were captured using laser microdissection and put into lysis buffer to obtain genomic DNA. Whole genomic DNA was bisulfite-converted using the EZ DNA Methylation-Direct Kit (Cat. D5021, ZYMO RESEARCH), and then amplified using the EpiTect Whole Bisulfitome Kit (Cat. 59203, QIAGEN). After PCR purification, a Roche 454 sequencer was used to assess the bisulfite sequencing. Associated software performed the alignment and mapping to the original sequence. Quantity of purified DNA after whole genomic amplification Initially, we aimed to verify that DNA methylation measurements could be reliably obtained from FFPE ovarian epithelial tissues. After bisulfite conversion, whole genomic amplification with (...truncated)