Improved amplification efficiency on stool samples by addition of spermidine and its use for non-invasive detection of colorectal cancer.

Jean-Pierre Roperch,Karim Benzekri,Roberto Incitti,Hicham Mansour

doi:10.1186/s12896-015-0148-6

Abstract

BackgroundUsing quantitative methylation-specific PCR (QM-MSP) is a promising method for colorectal cancer (CRC) diagnosis from stool samples. Difficulty in eliminating PCR inhibitors of this body fluid has been extensively reported. Here, spermidine is presented as PCR facilitator for the detection of stool DNA methylation biomarkers using QM-MSP. We examined its effectiveness with NPY, PENK and WIF1, three biomarkers which we have previously shown to be of relevance to CRC.ResultsWe determined an optimal window for the amplification of the albumin (Alb) gene (100 ng of bisulfite-treated stool DNA added of 1 mM spermidine) at which we report that spermidine acts as a PCR facilitator (AE = 1680%) for SG RT-PCR. We show that the amplification of methylated PENK, NPY and WIF1 is considerably facilitated by QM-MSP as measured by an increase of CMI (Cumulative Methylation Index, i.e. the sum of the three methylation values) by a factor of 1.5 to 23 fold in individual samples, and of 10 fold in a pool of five samples.ConclusionsWe contend that spermidine greatly reduces the problems of PCR inhibition in stool samples. This observed feature, after validation on a larger sampling, could be used in the development of stool-based CRC diagnosis tests.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-015-0148-6) contains supplementary material, which is available to authorized users.

Highlights

Using quantitative methylation-specific PCR (QM-MSP) is a promising method for colorectal cancer (CRC) diagnosis from stool samples
We observed that thymidine are detected instead of cytosine, as expected after DNA bisulfite modification of unmethylated amplicon products, since they correspond to a region of Alb which does not contain CpG sites
Those findings indicate that all cytosine are converted to thymine as a result of the DNA modification step being performed successfully and that the spermidine do not interferes in the specificity of PCR

Summary

Introduction

Using quantitative methylation-specific PCR (QM-MSP) is a promising method for colorectal cancer (CRC) diagnosis from stool samples. Difficulty in eliminating PCR inhibitors of this body fluid has been extensively reported. Spermidine is presented as PCR facilitator for the detection of stool DNA methylation biomarkers using QM-MSP. The development of CRC results from the progressive accumulation of genetic and epigenetic alterations leading to the transformation of normal colonic epithelium to colon adenocarcinoma [4,5,6]. Epigenetic alterations have been found frequently in neoplastic diseases [9,10]. It has been reported that the analysis of DNA methylation carried out in body fluids represents a valuable source for the discovery of cancer biomarkers [11].

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