Determination of Telomerase Activity in Tumors and Cancer Cell Lines
By PCR-ELISA
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The present study was undertaken to determine the presence of telomerase activity in normal and cancerous cell lines and tumor tissues by combined PCR-ELISA assay. Cell lines, canine mammary tumor tissues and human oral cancer tissues were processed for protein extraction. The concentration of the protein was determined by Bradford s method and used for Telomere Repeat Amplification Protocol (TRAP). The amplified products were electrophoresed on 20% non-denaturing polyacrylamide gel along with 10bp ladder. The presence of 6 base repeats starting from 50 nucleotides is considered as positive. T...
The present study was undertaken to determine the presence of telomerase activity in normal and cancerous cell lines and tumor tissues by combined PCR-ELISA assay. Cell lines, canine mammary tumor tissues and human oral cancer tissues were processed for protein extraction. The concentration of the protein was determined by Bradford s method and used for Telomere Repeat Amplification Protocol (TRAP). The amplified products were electrophoresed on 20% non-denaturing polyacrylamide gel along with 10bp ladder. The presence of 6 base repeats starting from 50 nucleotides is considered as positive. Then the TRAP products were immobilized on to streptavidin coated microtitre plates via biotin - streptavidin interaction and then detected by anti-DNP antibody conjugated with horse radish peroxidase. The absorbance was measured at 450 nm and at 690 nm. Samples was regarded as telomerase positive if A is greater than 0.2, where A: net increase of absorbance for the sample = A sample A heat-treated sample.Telomerase serves as an efficient biomarker for diagnosis of tumor and cell lines serves as a model system to investigate the efficacy of telomerase targeted therapeutics for cancer.
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