An automated high throughput solution for DNA extraction and bisulfite-conversion from high volume liquid biopsy specimens – sample preparation for epigenetic analysis

Abstract

Objective: DNA methylation analysis via real-time PCR or other analytical techniques requires purified bisulfite converted DNA. We report on an automated high throughput solution for DNA extraction, bisulfite-conversion, and purification of 96 samples with an input volume of up to 3.5mL of plasma or urine, using reagents from the commercially available Epi BisKit. Results: Magnetic bead-based DNA extraction, bisulfite conversion at high temperature, and efficient DNA purification was conducted on a customized commercially available liquid-handling platform. A highly interlaced 4x24 sample protocol was implemented for DNA extraction, elution in a 96-well plate, efficient bisulfite-conversion and extensive purification. The resulting bisulfite-converted DNA was stored in a 96-well format, ready for PCR set-up or other down-stream applications. The automated method is a walk-away solution for processing 96 samples in 7h30min. Performance of the method was validated by comparison with the standard manual method of the Epi BiSKit using technical and biological samples. Overall DNA yield was assessed with a standardized ß-actin assay. The automated workflow demonstrated equivalent performance to the manual method for technical, plasma and urine samples. It may provide a new standard for effective high-throughput preparation of bisulfite-converted DNA from a variety of high volume liquid biopsy specimens.