Microarray TRAP--a high-throughput assay to quantitate telomerase activity.
Telomeric repeat amplification protocol (TRAP)--a sensitive, PCR-based assay to detect telomerase activity was quintessential to the evaluation of telomerase role in telomere maintenance, cell proliferation, tumour development, and cell immortalization. The assay, however, suffers from many limitations. The most significant are: lack of telomerase activity quantification, changes of the enzyme activity product size and/or ratio, and complex post-amplification procedures which limit the assay throughput. Here we report the development of the microarray TRAP (MTRAP) assay which combines advantages of microarray technology with a modified TRAP assay. The MTRAP was designed and optimized on rice cell suspension telomerase extract to enable telomerase specific, reliable, and linear quantification in high throughput mode, with sensitivity comparable to those of radioisotope-based TRAP assays. The MTRAP has a built-in system guaranteeing the amplification of telomerase activity products unchanged in length and/or ratio and built-in control for false negatives. Thus, our MTRAP assay provides new reliable tool for experiments requiring massive quantitation of telomerase activity.
Heller-Uszynska
Katarzyna
K
CAMBIA: Center for the Application of Molecular Biology to International Agriculture, G.P.O. Box 3200, Canberra, ACT 2601, Australia.
Kilian
Andrzej
A
D004789:Enzyme Activation; D020869:Gene Expression Profiling / Q000379:methods*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D012275:Oryza / Q000201:enzymology* / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019098:Telomerase / Q000032:analysis* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D019098:Telomerase