The telomerase repeat amplification protocol (TRAP) assay, first described by Kim et al. (1994), relies upon primer extension of an oligonucletide by telomerase. Cells are lysed and cellular protein extracts are incubated with an oligonucletide to which a series of TTAGGG repeats will be added when telomerase is present in the cell extract. Variations of the TRAP assay exist, including radioactive or nonradioactive gel-based detection, ELISA-based detection and semiquantitative or quantitative protocols. For an excellent review of the TRAP assay and many of its iterations, see Saldanha et al. (2003).
GENE EXPRESSION ANALYSIS Real-time fluorescence-based PCR and RT-PCR have emerged as powerful methods for examining gene expression patterns in many contexts. In traditional PCR, an amplicon which accumulates after a predetermined number of cycles is analyzed by gel electrophoresis. In real-time PCR, reactions are characterized by the PCR cycle at which amplification of a target molecule is first detected by release of a fluorescent signal in real time. The greater the quantity of the target molecule in the reaction mix, the earlier a significant increase in fluorescence will be measured. Quantitation is accomplished with reference to a threshold cycle, (Ct), defined as the fractional cycle number at which fluorescence, generated by the increase in PCR product, exceeds a set threshold above the baseline. For an excellent treatise on fluorescence-based real-time PCR, refer to Bustin A-Z of Quantitative PCR (2004).
Recently, real-time quantitative TaqMan PCR was utilized to look at expression of genes involved in chicken telomere maintenance pathways. Chicken primers and fluorescent probes were developed for seven target genes (tankyrase 1, tankyrase 2, TRF1, TRF2, cTERT, cTR and c-myc) as well as for three housekeeping genes for normalization purposes. In cell culture, chicken GAPDH mRNA levels were found to show the least standard deviation for all samples examined, and therefore GAPDH values were used to normalize the target gene values.
Analysis of mRNA expression patterns of the target genes in CEFs, DT40, the gastrula embryo and cES cells revealed up-regulation of tankyrase 2, TRF1, TRF2, c-myc, cTERT and cTR in DT40 cells, with c-myc levels up-regulated 184-fold in DT40 relative to the gastrula and 282-fold in DT40 relative to CEFs and cES cells. Telomerase holoenzyme components (cTERT and cTR) were present, although at low levels, in CEFs and were up-regulated in DT40, cES cells and the gastrula relative to CEFs. Down-regulation of TRF1, c-myc, cTERT and cTR appeared to be a feature of senescing CEFs that had survived an average of 30.5 PD (Swanberg et al., 2004; Swanberg and Delany, 2005). For a detailed discussion of these expression patterns as well as primer and probe sets, for target and housekeeping genes, see Swanberg et al. (2004) and Swanberg and Delany (2005).
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