Quantitative Analysis Of Epidermal Growth Factor Receptor MRNA In Laryngeal Carcinoma By Reverse Transcription-Real Time Polymerase Chain Reaction With TagMan Probe

The overexpression and inappropriate activation of epidermal growth factor receptor (EGFR) is thought to play an important role in many tumors, especially for the head and neck carcinomas with the overexpression rate of 80%—100%.So far there have been many articles on the expression of EGFR in laryngeal carcinomas. Most of them have confirmed its overexpression, but there still remained some controversies. The immunohistochemistry and traditional reverse transcription polymerase chain reaction (RT—PCR) have been commonly used in the quantitive analysis of EGFR for the convenience previously, however their shortcomings are also obvious. We suppose that different outcomes on the expression of EGFR are probably due to the lack of an objective technology with higher accuracy in the quantitive evaluation of EGFR in laryngeal carcinomas.The technology of reverse transcription-real time polymerase chain reaction with TagMan probe is now widely used in gene quantitation as it is highly sequence-specific, more sensitiveand amenable to increasing sample throughput with stable outcomes when compared with some traditional methods of mRNA quantitation, namely RT-PCR or Northern blot. In this paper we reported the application of reverse transcription-real time polymerase chain reaction with TagMan probe for EGFR mRNA quantitation in laryngeal carcinoma tissues.Materials and Method 1. Clinical MaterialsTumor tissues and macroscopically normal laryngeal mucosas adjacent to the tumor were collecteded from 32 patients with primary laryngeal carcinoma admitted to our department without any treatment before from June 2003 to September 2005. All these 32 cases were male , with the median age of 57. 5. The specimens were taken immediately after removal in operation room, kept in liquid nitrogen and then stored at -80℃ until they were analyzed. The diagnosis of all the cases was confirmed pathologically. 32 cases of laryngeal carcinomas were distinguished into well differentiated (4), moderately differentiated (19) and poorly differentiated (9) squamous cell carcinomas. According to the TNM stage stipulted by UICC and AJCC in 2002, these 32 cases of laryngeal carcinomas were classified as stage I (5), stage II (12), stage III (8) and stage IV (7). 2. MethodThe primers and probe for EGFR was designed by Primer Express Software v2.0. The recombinant plasmid of pGEM-T Easy /EGFR was constructed , and the gradient dilutedplasmid was formed after comfirmation of EGFR by sequence analysis.All the tissue samples underwent the RNA extraction and cDNA synthesis by a routine way. Then the detection of real-time PCR with TagMan probe was performed to detection the expression of EGFR mRNA, using GAPDH as its internal control. 3. Data analysisThe relative amount of EGFR mRNA was equal to 2^-ΔCT (ΔCT=CT_EGFR—CT_GAPDH. The acquired data was analyzed by SPSS 12.0 software.According to one sample kdmogorov-smimor test, it represented a skewness distribution. So the results were expressed in the form of median (interquartile range).The differences were statistically analyzed with the rank sum Wilcoxon test.ResultsGene sequencing comfirmed that the recombinant plasmid of pGEM-T Easy/EGFR was successfully cloned. And the method of real-time PCR with TagMan probe was also successfully constructed for the quantitative detection of EGFR mRNA.The median of the relative content of EGFR mRNA in Iaryngeal carcinomas and macroscopically normal laryngeal mucosas adjacent to the tumors were shown in Table 1-1 .The difference between them was statistically significant in Wilcoxon Rank test (P<0.01).Table 1 -1. Relative content of EGFR mRNA in laryngeal carcinoma tissues and macroscopically normal laryngeal mucosas adjacent to the tumorConclusion1. The mRNA index of EGFR in laryngeal carcinoma tissues was significantly higher than that in their own macroscopically normal laryngeal mucosas adjacent to the tumors. The amplification of EGFR mRNA would probably play an important role in the up-regulation of EGFR.2. The method of reverse transcription-real time polymerase chain reaction with TagMan probe is quite objective, precise and high throughput in the application for the detection of EGFR mRNA in laryngeal carcinomas.