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[Construction of a t7 human lung cancer cDNA library.]
Wentao Yue1, Zitong Wang, Yue Wang
1Department of Cellular and Molecular Biology, Beijing TB and Thoracic Tumor Research Institute, Beijing 101149, China. yuewt2000@yahoo.com.
Background:
Currently, only a limited numbers of tumor markers for non small lung cancer (NSCLC) diagnosis, new biomarker, such as serum autoantibody may improve the early detection of lung cancer. Our objective is construction human lung squamous carcinoma and adenocarcinoma T7 phage display cDNA library from the tissues of NSCLC patients.
Methods:
mRNA was isolated from a pool of total RNA extract from NSCLC tissues obtained from 5 adenocarcinomas and 5 squamous carcinomas, and then mRNA was reverse transcribed into double stranded cDNA. After digestion, the cDNA was inserted into T7Select 10-3 vector. The phage display cDNA library was constructed by package reaction in vitro and plate proliferation. Plaque assay and PCR were used to evaluate the library.
Results:
Two T7 phage display cDNA library were established. Plaque assay show the titer of lung squamas carcinoma library was 1.8*10(6) pfu, and the adenocarcinoma library was 5*10(6) pfu. The phage titer of the amplified library were 3.2*10(10) pfu/mL and 2.5*10(10) pfu/mL. PCR amplification of random plaque show insert ratio were 100% (24/24) in adenocarcinoma library and 95.8% in human lung squamas carcinoma library (23/24). Insert range from 300 bp to 1 500 bp.
Conclusions:
Two phage display cDNA library from NSCLC were constructed.
