Digital analysis of the expression levels of multiple colorectal cancer-related genes by multiplexed digital-PCR coupled with hydrogel bead-array

摘要

To digitally analyze expression levels of multiple genes in one reaction, we proposed a method termednas ‘MDHB’ (Multiplexed Digital-PCR coupled with Hydrogel Bead-array). The template for beadbasednemulsion PCR (emPCR) was prepared by reverse transcription using sequence-tagged primers.nThe beads recovered from emPCR were immobilized with hydrogel to form a single-bead layer onna chip, and then decoded by gene-specific probe hybridization and Cy3-dUTP based primer extensionnreaction. The specificity of probe hybridization was improved by using electrophoresis to removenmismatched probes on the bead’s surface. The number of positive beads reflects the abundance ofnexpressed genes; the expression levels of target genes were normalized to a housekeeping gene andnexpressed as the number ratio of green beads to red beads. The discrimination limit of MDHB is 0.1%n(i.e., one target molecule from 1000 background molecules), and the sensitivity of the method is belown100 cells when using the b-actin gene as the detection target. We have successfully employed MDHB tondetect the relative expression levels of four colorectal cancer (CRC)-related genes (c-myc, COX-2,nMMP7, and DPEP1) in 8 tissue samples and 9 stool samples from CRC patients, giving the detectionnrates of 100% and 77%, respectively. The results suggest that MDHB could be a potential tool for earlynnon-invasive diagnosis of CRC.