cDNA Library Construction and Confirmation of Human Tongue Squamous Cell Carcinoma

Objective: In order to clone and screen those proliferation/differentiation related genes and establish technique platform for yeast two hybrid system, we chose the established human tongue squamous cell carcinoma(Tca8113) cell line to construct the cDNA library of oral squamous cell carcinoma. Methods: Yeast two hybrid construction and screening kit of Clontech® were chosen to construct this cDNA library. Expriments were processed as followings: Total RNA of Tca8113 was extracted and purified to mRNA with Qiagen mRNA purify kit, whose quality was examined with electropheresis. Then, mRNA was further reverse-transcribed into cDNA, and the sequences of smart III and CDS III was integrated into its 3' end and 5' end at the same time. LD-PCR was processed with special primers, and examined its products with electrophersis before purify. The purified products and linearized pGADT7-Rec was contransformed into the competent AH109. After growing on SD/-Leu plate till colonies appear, these transformants were harvest and frozen at -80°C. 10 clones were randomly picked out to test the quality of this library. Results: Products of LD-PCR were smearing bands, size from 500bp-3kb. Calculate the transformation efficiency of this library by counting the colonies growing on the SD/-Leu plate, which was 1°Á106 transformants / 3µg pGADT7-Rec. To determine the library size, spread 1:100, 1:1000, 1:10, 000 dilution on SD/-Leu plates, and count the colonies (cfu), calculate the number of colonies in library as 4°Á107cfu/ml. The size of PCR products of these ten randomly picked clones were from 500bp to 3kb. Conclusions: We constructed a cDNA library of human squamous cell carcinoma cell line(Tca8113) with yeast two hybrid construction and screening kit and examined its quality, which could be used to further screen and clone those etiology related genes in oral squamous cell carcinoma.