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

Li, Qiling; Li, Min; Ma, Li; Li, Wenzhi; Wu, Xuehong; Richards, Jendai; Fu, Guoxing; Xu, Wei; Bythwood, Tameka; Xu, Li; Wang, Jianxin; Song, Qing

doi:10.1371/journal.pone.0104481

Cited by 11 publications

(9 citation statements)

References 44 publications

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“…Due to DNA degradation and fragmentation, the mapping rates for FFPE samples are lower than those that would be expected from fresh tissue or cell line material. For example, a previous genome-wide evaluation of FFPE material reported unique mapping rates of 7.0% to 19.9% [ 16 ]. Following the protocol described here, we obtained a unique mapping efficiency of 35–40% (Data S1 and S2 in Additional file 1 ).…”

Section: Discussionmentioning

confidence: 99%

A streamlined method for analysing genome-wide DNA methylation patterns from low amounts of FFPE DNA

et al. 2017

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Background: Formalin fixed paraffin embedded (FFPE) tumor samples are a major source of DNA from patients in cancer research. However, FFPE is a challenging material to work with due to macromolecular fragmentation and nucleic acid crosslinking. FFPE tissue particularly possesses challenges for methylation analysis and for preparing sequencing-based libraries relying on bisulfite conversion. Successful bisulfite conversion is a key requirement for sequencing-based methylation analysis. Methods: Here we describe a complete and streamlined workflow for preparing next generation sequencing libraries for methylation analysis from FFPE tissues. This includes, counting cells from FFPE blocks and extracting DNA from FFPE slides, testing bisulfite conversion efficiency with a polymerase chain reaction (PCR) based test, preparing reduced representation bisulfite sequencing libraries and massively parallel sequencing. Results: The main features and advantages of this protocol are:

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Section: Discussionmentioning

confidence: 99%

A streamlined method for analysing genome-wide DNA methylation patterns from low amounts of FFPE DNA

et al. 2017

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“…The nested PCRs used to amplify the E2BS1 and E2BS2,3,4/Sp1 binding sites were more sensitive than the primary PCRs alone, allowing methylation analysis in more than 80% of the samples. This higher level of detection may be in part due to the double amplification round, the smaller amplified product generated in the nested PCRs compared to the amplified products of the primary PCRs, and to a potential increase in efficiency of PCR amplification during the nested PCRs due to increased dilution of possible PCR inhibitors [ 31 – 32 ]. Reports from other researchers have indicated limited success analysing HPV methylation using LCM tissues, due to limited sensitivity of the assays used [ 20 ].…”

Section: Discussionmentioning

confidence: 99%

CpG Methylation Analysis of HPV16 in Laser Capture Microdissected Archival Tissue and Whole Tissue Sections from High Grade Anal Squamous Intraepithelial Lesions: A Potential Disease Biomarker

et al. 2016

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Incidence and mortality rates of anal cancer are increasing globally. More than 90% of anal squamous cell carcinomas (ASCC) are associated with human papillomavirus (HPV). Studies on HPV-related anogenital lesions have shown that patterns of methylation of viral and cellular DNA targets could potentially be developed as disease biomarkers. Lesion-specific DNA isolated from formalin-fixed paraffin-embedded (FFPE) tissues from existing or prospective patient cohorts may constitute a valuable resource for methylation analysis. However, low concentrations of DNA make these samples technically challenging to analyse using existing methods. We therefore set out to develop a sensitive and reproducible nested PCR-pyrosequencing based method to accurately quantify methylation at 10 CpG sites within the E2BS1, E2BS2,3,4 and Sp1 binding sites in the viral upstream regulatory region of HPV16 genome. Methylation analyses using primary and nested PCR-pyrosequencing on 52 FFPE tissue [26 paired whole tissue sections (WTS) and laser capture microdissected (LCM) tissues] from patients with anal squamous intraepithelial lesions was performed. Using nested PCR, methylation results were obtained for the E2BS1, E2BS2,3,4 and Sp1 binding sites in 86.4% of the WTS and 81.8% of the LCM samples. Methylation patterns were strongly correlated within median values of matched pairs of WTS and LCM sections, but overall methylation was higher in LCM samples at different CpG sites. High grade lesions showed low methylation levels in the E2BS1 and E2BS2 regions, with increased methylation detected in the E2BS,3,4/Sp1 regions, showing the highest methylation at CpG site 37. The method developed is highly sensitive in samples with low amounts of DNA and demonstrated to be suitable for archival samples. Our data shows a possible role of specific methylation in the HPV16 URR for detection of HSIL.

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“…The slides were dried in an incubator at 37°C for 3 hours. Hematoxylin and eosin staining was performed as discussed elsewhere [ 48 ]. Briefly, the slides were rehydrated, stained, and dehydrated, followed by drying for 1 hour at room temperature.…”

Section: Methodsmentioning

confidence: 99%

Klotho inhibits EGF-induced cell migration in Caki-1 cells through inactivation of EGFR and p38 MAPK signaling pathways

et al. 2018

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Klotho is a single-pass transmembrane protein with documented anti-cancer properties. Recent reports have implicated Klotho as an inhibitor of transforming growth factor β1 induced cell migration in renal fibrosis. Overexpression of epidermal growth factor receptor (EGFR) is known to promote tumor initiation and progression in clear-cell renal cell carcinoma (cRCC). We tested our hypothesis that Klotho inhibits EGF-mediated cell migration in cRCC by interfering with the EGFR signaling complex and mitogen-activated protein kinase (MAPK) pathways. We performed cell adhesion, migration, and biochemical studies in vitro using Caki-1 cell line. In addition, we validated the cell culture studies with expression analysis of six de-identified FFPE tissues from primary and metastatic cRCC patients. Our studies show that Klotho inhibited EGF-induced Caki-1 de-adhesion and decreased spreading on collagen type 1. Klotho also inhibited EGF-induced α2β1 integrin-dependent cell migration on collagen type 1. To test the involvement of MAPK pathways in EGF-induced Caki-1 cell motility, the cells were pretreated with either SB203580, a specific p38 MAPK inhibitor, or Klotho. SB203580 blocked the EGF-induced Caki-1 cell migration. Klotho had a comparable inhibitory effect. Our FFPE clinical specimens revealed decreased Klotho mRNA expression compared to a control, non-cancer kidney tissue. The decrease in Klotho mRNA levels correlated with increased c-Src expression, while E-Cadherin was relatively reduced in metastatic FFPE specimens where Klotho was least expressed. Taken together, these results suggest that secreted Klotho inhibits EGF-induced pro-migratory cell morphological changes and migration in Caki-1 cells. Our data additionally suggest that decreased Klotho expression may be involved in cRCC metastasis.

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

Cited by 11 publications

References 44 publications

A streamlined method for analysing genome-wide DNA methylation patterns from low amounts of FFPE DNA

A streamlined method for analysing genome-wide DNA methylation patterns from low amounts of FFPE DNA

CpG Methylation Analysis of HPV16 in Laser Capture Microdissected Archival Tissue and Whole Tissue Sections from High Grade Anal Squamous Intraepithelial Lesions: A Potential Disease Biomarker

Klotho inhibits EGF-induced cell migration in Caki-1 cells through inactivation of EGFR and p38 MAPK signaling pathways

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