Telomerase stability and evaluation of real-time telomeric repeat amplification protocol

Abstract

Telomerase is RNA directed polymerase which acts as reverse transcriptase based on its own RNA component. It is considered to be involved in the pathology of many diseases and is recognized as a potential biomarker. The aims were to determine the sample storage conditions and the time frame for samples analysis, then to prove reliability of enzyme activity measurement with real-time telomeric repeat amplification protocol (TRAP) and to evaluate the suitable standard samples for telomerase activity measurements. Samples used for stability and freeze-thaw study were peripheral blood leukocytes, obtained from apparently healthy persons, patients with diagnosed cancer and cell lines. Telomerase activity was measured using TRAP method, while standard evaluation was done using nuclear magnetic resonance (NMR) technique. Storage at −20 °C preserved telomerase activity in samples from cancer patients for at least 14 days (21.46 ± 0.135 versus 21.84 ± 0.357, p = .756), while samples obtained from healthy persons should be stored at −80 °C. We observed significant decrease of telomerase activity at freeze thaw cycle 5 in cancer patients’ samples (21.46 ± 0.135 versus 23.09 ± 0.316, p < .05), and in healthy persons’ ones already at cycle 3 (22.74 ± 0.107 versus 24.85 ± 0.151, p < .05). Telomerase activity from cell lines samples showed overall greater stability regarding the storage period and freeze-thaw cycles and it was considered for standard sample, which was confirmed by NMR analysis. Telomerase enzyme had adequate stability while efficacy, linearity, and reproducibility of TRAP method were acceptable for bio-analytical methods. All this indicated that telomerase could be a reliable biomarker.

Keywords:

• Telomerase
• freezing
• reproducibility of results
• biomarkers
• carcinoma
• nuclear magnetic resonance

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work was supported by the by the Ministry of Education, Science and Technological Development, Republic of Serbia [Grant no. 175035] and Slovenian Research Agency [Grants P3-0298 and J3-5511].