Construction of the method for quantitative detection of epidermal growth factor receptor variant Ⅲ by real-time polymerase chain reaction with Taq Man probe and its application
10.3321/j.issn:1673-0860.2008.04.012
- VernacularTitle:实时荧光定量聚合酶链反应测定表皮生长因子Ⅲ突变体表达的方法构建及其应用
- Author:
Bei-Bei YANG
1
;
Xing ZHANG
;
Jia CHEN
;
Jiang CAO
;
Hui-Yu CHANG
Author Information
1. 浙江大学医学院附属第二医院
- Keywords:
Receptor,epidermal growth factor;
Plasmids;
Polymerase chain reaction;
Laryngeal neoplasmas;
Carcinoma,squamous cell
- From:
Chinese Journal of Otorhinolaryngology Head and Neck Surgery
2008;43(4):296-301
- CountryChina
- Language:Chinese
-
Abstract:
Objecfive To construct the recombinant plasmid and standard curve for detection of epidermal growth factor receptor variant Ⅲ (EGFRv Ⅲ) by real-time quantitive polymerase chain reaction (PCR) and establish the RT-PCR assay for accurate detection of EGFRv Ⅲ.Methods To amplify the DNA sequences of exon 1 and exon 8 to exon 9 of EGFR seperately and fuse the above sequences by overhang extension PCR.Then constructing the recombinant plasmid of EGFRv Ⅲ by highly specific primers with the fused sequence as its template.The recombinant plasmid of EGFRv Ⅲ was then sent for sequence analysis.The gradient diluted recombinant plasmid were used as the template to amplify the EGFRv Ⅲ by RT-PCR with TaqMan probe and then applied it in 32 laryngeal carcinoma tissues.Results The recombinant plasmid of EGFRv Ⅲ was successfully constructed.The gradient diluted recombinant plasmid could be used in RT-PCR for quantitative analysis of EGFRv Ⅲ.In all,5 tumor samples expressing EGFRv Ⅲ were detected,with its positive rate of about 15.6%.The expression of EGFRv Ⅲ was tumor specific with only one exception for extraordinarily low expression of EGFRv Ⅲ in macroscopically normal laryngeal mucosas adiacent to the tumor.Conclusions RT-PCR with TaqMan probe was good at sensitivity,specificity,quantification and linear function.It can be a standard method for quantitative detection for EGFRv Ⅲ.Expression of EGFRv Ⅲ was detected in laryngeal carconoma tissues with relatively low level.Whether EGFRv Ⅲ was a suitable target for biological therapy in laryngeal carcinomas remained to be discussed.
