The adenomatous polyposis coli (APC) gene has been shown to be involved in genetic instability and to be downregluated in several human carcinomas. The chromosome locus of APC, 5q21-22, is frequently deleted in colorectal cancers (CRCs). The functional impact of such regions needs to be extensively investigated in large amount of clinical samples. Case-matched tissues of CRC and adjacent normal epithelium (n = 134) were included in this study. Quantitative PCR was carried out to examine the copy number as well as mRNA expression of APC gene in colorectal malignancies. Our results showed that copy number deletions of APC were present in a relatively high percentage of colorectal cancer samples (26.1%, 35 out of 134). There was a positive correlation between copy number decrease of APC and tumor progression in CRCs. Furthermore, copy number loss of APC was correlated with decreased mRNA expression. However, mRNA levels of APC were also impaired in CRC samples with unaltered copy numbers, indicating that sporadic CRCs exhibit different mechanisms of APC regulation.
Colorectal cancerAPCCopy number variationGene expression
The representative diagram of the DNA CNV analysis for one cancer/ANT pair. (A) Real-time PCR amplification of targeted gene in selected genomic DNA samples. Each data was obtained from two independent reactions. (B) Original Ct values obtained from the real-time PCR amplification. (C&D) The efficiency of and slope of the RNAse P and APC amplification were calculated by Bio-Rad Thermal Cyclers software. The detailed calculation was performed as follow: dCt = average Ct (APC)-average Ct (RNAse P); ddCt (sample1) = dCt (CRC)- dCT (ANT) = 2.96; Etarget was determined by the efficiency of target gene amplification. Cut-off value (sample1) = E−ddCt = 2.05−2.96 = 0.12. The copy number of APC in sample1 is 0. (GIF 24 kb)
The representative diagram of the calculation of mRNA expression for one cancer/ANT pair. (A) Real-time PCR amplification of targeted gene in selected cDNA samples. Each data was obtained from two independent reactions. (B) Original Ct values obtained from the real-time PCR amplification. (C&D) The efficiency of and slope of the GAPDH and APC amplification were calculated by Bio-Rad Thermal Cyclers software. 2−∆Ct method was used to calculate the obtained results, and the mRNA level of APC was expressed as a ratio relative to the GAPDH mRNA in each sample. For this sample: ∆Ct (CRC) = average Ct (APCCRC)-average Ct (GAPDHCRC) =5.59; ratio (CRC) = 2−∆Ct(CRC) = 0.021 = 2.1%; ∆Ct (ANT) = average Ct (APCANT)-average Ct (GAPDHANT) =2.88; ratio (ANT) =2−∆Ct(ANT) =0.136 = 13.6%. (GIF 24 kb)