Different Genes CDNA Libraries Of High And Low Aldehyde Dehydrogenase Activity Cells Of Tongue Squamous Carcinoma Were Constructed

Objective To construct differentially expressed cDNA libraries withhigh and low aldehyde dehydrogenase activity(ALDH^lowhigh/ALDH^lowlow) cells oftongue squamous carcinoma Tca8113cell line. Methods Expression ofstem cell marker ALDH^low was detected,and ALDH^lowhighand ALDH^lowlowcellswere collected by Aldefluor assay combined with the flow cytometry;Differentially expressed genes of total RNA that was extracted from thetwo cell subpopulations by Trizol were screened and amplified bysuppressing subtractive hybridization（SSH）,and the PCR products wereconnected with pMD18-T Vector, and then transfected into E.coli DH5α foramplification.24positive clones randomly picked from each library weredigested， sequenced and homologously analyzed. Results Twosubpopulations of ALDH^lowhighå'ŒALDH^lowlowwere screened out,and ALDH^lowhighcells in Tca8113cell line accounted for2.5%; RNA D（260）/D（280） ofALDH^lowhighå'ŒALDH^lowlowwere1.93and1.92.Two-directional subtractivelibraries of ALDH^lowhighå'ŒALDH^lowlowwere constructed,and each librarycomprised500clones, PCR analysis of24clones randomly picked from each library showed that insert-fragments distributed in200-700bp,and nofalse positive clones were detected. Gene sequencing result that washomologously analyzed and indexed by PubMed showed that cancer relatedgenes included SLC25A13, KLHL2, NPC1, WAPL, BARD1, Notch2andEEF2K. Conclusion Two-directional subtractive cDNA libraries ofALDH^lowhighand ALDH^lowlowcells were successfully constructed.