Quality assessment of DNA derived from up to 30 years old formalin fixed paraffin embedded (FFPE) tissue for PCR-based methylation analysis using SMART-MSP and MS-HRM

Abstract

Background: The High Resolution Melting (HRM) technology has recently been introduced as a rapid and robust analysis tool for the detection of DNA methylation. The methylation status of multiple tumor suppressor genes may serve as biomarkers for early cancer diagnostics, for prediction of prognosis and for prediction of response to treatment. Therefore, it is important that methodologies for detection of DNA methylation continue to evolve. Sensitive Melting Analysis after Real Time - Methylation Specific PCR (SMART-MSP) and Methylation Sensitive - High Resolution Melting (MS-HRM) are two methods for single locus DNA methylation detection based on HRM.

Methods: Here, we have assessed the quality of DNA extracted from up to 30 years old Formalin Fixed Paraffin Embedded (FFPE) tissue for DNA methylation analysis using SMART-MSP and MS-HRM. The quality assessment was performed on DNA extracted from 54 Non-Small Cell Lung Cancer (NSCLC) samples derived from FFPE tissue, collected over 30 years and grouped into five years intervals. For each sample, the methylation levels of the CDKN2A (p16) and RARB promoters were estimated using SMART-MSP and MS-HRM assays designed to assess the methylation status of the same CpG positions. This allowed for a direct comparison of the methylation levels estimated by the two methods for each sample.

Results: CDKN2A promoter methylation levels were successfully determined by SMART-MSP and MS-HRM in all 54 samples. Identical methylation estimates were obtained by the two methods in 46 of the samples. The methylation levels of the RARB promoter were successfully determined by SMART-MSP in all samples. When using MS-HRM to assess RARB methylation five samples failed to amplify and 15 samples showed a melting profile characteristic for heterogeneous methylation. Twenty-seven of the remaining 34 samples, for which the methylation level could be estimated, gave the same result as observed when using SMART-MSP.

Conclusion: MS-HRM and SMART-MSP can be successfully used for single locus methylation studies using DNA derived from up to 30 years old FFPE tissue. Furthermore, it can be expected that MS-HRM and SMART-MSP will provide similar methylation estimates when assays are designed to analyze the same CpG positions.

Figure 1

The design of the CDKN2A and RARB SMART-MSP and MS-HRM assays. The SMART-MSP assays were designed to analyze the same CpG positions as the MS-HRM assays to allow for a direct comparison of the methylation levels in the samples. The MS-HRM primers are denoted as green arrows and the SMART-MSP primers as orange arrows. The CpG positions are denoted as lollipops.

Figure 2

The sensitivity of the CDKN2A and RARB SMART-MSP and MS-HRM assays. The sensitivity of the assays was tested using standard dilution series of methylated DNA into unmethylated DNA. The 100% methylated standards are indicated in red, the 10% methylated standards in blue, the 1% methylated standards in green, the 0.1% methylated standards in brown, the 0% methylated standard in orange and non template controls are indicated in black. A. The CDKN2A SMART-MSP assay (real-time amplification data). B. The CDKN2A MS-HRM assay (normalized melting curves). C. The RARB SMART-MSP assay (real-time amplification data). D. The RARB MS-HRM assay (normalized melting curves). All assays successfully detect 0.1% methylation.

Figure 3

Melting profiles indicating that the DNA is heterogeneously methylated at the RARB promoter. If molecules, in which different CpG positions are methylated and unmethylated are amplified, the observed melting profile will be complex. These different molecules will form heteroduplexes and homoduplexes after the PCR and prior to the HRM step. The very early melting observed is likely to be the melting of various heteroduplexes. The intermediate and later melting are likely to be the melting of various homoduplexes with those having the highest number of CpG positions methylated melting the latest. The 100% methylated standards are indicated in red, the 10% methylated standards in blue, the 1% methylated standards in green, the 0.1% methylated standards in brown, the 0% methylated standard in orange and non template controls are indicated in black. A sample (in triplicate) judged to be heterogeneously methylated is indicated in turquoise.

Figure 4

Reproducibility of the methylation estimates for the FFPE samples by SMART-MSP. Plots of the absolute difference between the highest and lowest possible methylation estimate divided by the average methylation estimate against the age of the samples for each gene are shown. A. The CDKN2A assay. B. The RARB assay. It is observed that the overall reproducibility is better for the CDKN2A assay compared to the RARB assay. It can also be observed that the reproducibility did not depend on the age of the samples.

Figure 5

CDKN2A (p16) expression in NSCLC. A. A case in which immunohistochemical staining for p16 showed positive nuclear and cytoplasmic staining (×100). B. A case in which the invading tumor cells did not express p16 (×100).