In Situ Sodium Bisulfite Modification of Genomic DNA from Microdissected Melanoma Paraffin-Embedded Archival Tissues

Abstract

DNA methylation analysis of paraffin-embedded archival tumor tissues (PEAT) is important in clinical and translational research studies. Efficient identification and isolation of homogeneous cell population, optimal DNA extraction, and sodium bisulfite modification (SBM) are essential, particularly in small tumor lesions. Laser capture microdissection (LCM) coupled with an in situ SBM improves the specificity, through histopathology accuracy, and the amount of genomic DNA modified for downstream methylation assays.

Keywords: DNA methylation; Laser capture microdissection (LCM); Melanoma; Sodium bisulfite modification (SBM).

Figure 1

In situ-SBM algorithm. (a) Positioning of LCM-cap on tissue section. (b) Selection of cells of interest and pulse of infrared laser. (c) Separation of cells bounded to the LCM-cap polymer. Light micrographs show melanoma nested in heterogeneous tissue prior LCM (d), after LCM (e) and the LCM-cap containing the adhered targeted melanoma tissue sections (f). Black arrowheads indicate the targeted melanoma tissue. (g) Diagram of a microcentrifuge tube containing the LCM-cap with the isolated population of cells. (h) CFX manager 2.0 output view of the quantitative PCR amplification for methylated (M) and unmethylated (U) alleles.