Aim:
We recently identified that gene amplified in esophageal cancer 1 (GAEC1) shows different level of DNA copy numbers in non-neoplastic tissue and carcinoma which indicates its potential role in colorectal cancer (CRC) pathogenesis. This study aims to accurately quantify GAEC1 copy number variation (CNV) in colorectal cancer through the development of Evagreen multiplex assay in droplet digital PCR (ddPCR).
Methodology:
Fresh-frozen CRC tissues and matched adjacent non-tumour tissues near the surgical resection margin were collected from 80 Australian patients. Each tissue was pathologically verified and the clinicopathological details of the patient was noted. Genomic DNA was extracted from these samples and subjected to droplet digital (dd) PCR analysis. Primer sets concentration, sample input concentration and amplicon sizes were manipulated to enable maximum separation of fluorescence amplitude in both 1D and 2D droplet plots of QuantaSoft software version 1.7.4.
Results:
ddPCR duplex EvaGreen assay was successfully developed for the detection of GAEC1 CNV. We were able to determine GAEC1 copy number in 97% of the colorectal cancer cases. 75% (60/80) of them showed similar copy number when compared to their matched normal tissues. Of the 20 samples that showed variation, 18 samples showed GAEC1 amplification while only two samples showed GAEC1 deletion.
Conclusion:
The present results showed concordance between our previous published real-time PCR studies. Therefore, reliable detection of GAEC1 copy number alteration can be achieved with the use of ddPCR in a cost effective manner. This can help save tremendous cost if applied in future diagnostics and prognostic settings.