Telomere Length and Telomerase Activity; A Yin and Yang of Cell Senescence

Summary

An accurate, short, sophisticated and cheap method is described that assesses telomere length in multiple tissues and species using qRT-PCR. In addition, we will describe a simple assay to assess telomerase activity as a complementary backbone test for telomere length.

Abstract

Telomeres are repeating DNA sequences at the tip ends of the chromosomes that are diverse in length and in humans can reach a length of 15,000 base pairs. The telomere serves as a bioprotective mechanism of chromosome attrition at each cell division. At a certain length, telomeres become too short to allow replication, a process that may lead to chromosome instability or cell death. Telomere length is regulated by two opposing mechanisms: attrition and elongation. Attrition occurs as each cell divides. In contrast, elongation is partially modulated by the enzyme telomerase, which adds repeating sequences to the ends of the chromosomes. In this way, telomerase could possibly reverse an aging mechanism and rejuvenates cell viability. These are crucial elements in maintaining cell life and are used to assess cellular aging. In this manuscript we will describe an accurate, short, sophisticated and cheap method to assess telomere length in multiple tissues and species. This method takes advantage of two key elements, the tandem repeat of the telomere sequence and the sensitivity of the qRT-PCR to detect differential copy numbers of tested samples. In addition, we will describe a simple assay to assess telomerase activity as a complementary backbone test for telomere length.

Introduction

Telomeres are repeating DNA hexamer (TTAGGG) sequences found at the ends of chromosomes. In each cell replication, these chromosome ends are shortened. If they become too short, chromosomes can undergo telomere end fusions, aberrant recombination, and degradation. Thus sufficient telomere length maintenance plays a major role in chromosome stability and cell protection ³⁸. Telomere length maintenance is also crucial for genes found near the ends of chromosomes, since DNA replication cannot continue to the very end of chromosomes ^1-2. Consequently, prevention of telomere shortening may improve cell stability.

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Protocol

1. Telomere Length Protocol¹⁹

1. DNA isolation

Most DNA isolation methods may be used. Our Lab prefers Qiagen DNeasy Kit (#69506) for blood sources. Depending on the source of DNA, such as buccal or other sources, a different isolation method may be used.

2. Primers

All primers are diluted to a stock concentration of 100pmoles/μl in PCR grade water and stored at -20 °C until required. Working stocks of primers are made fresh and are stored at 20 °C for a short period of time (A few months.) Standard primers were used for telomeres and β-Gl....

Results

An example of a telomere length qRT-PCR assay is shown in Figure 1. On the top left panel, samples labeled in red and green represent the location of the tested subjects on the 96-well plate. On the top right panel, the amplification curve is demonstrated. Each subject is tested by two assays (Telomere and Single Copy Gene), which is done in triplicates. Due to different DNA copy numbers produced in the two assays of selected individuals, the number of cycles until the florescence detection reache.......

Discussion

Telomere length provides a unique cellular marker to study the stressed and aging cell and offers insights into the mechanisms of aging. Since its role in aging had first been suggested, a multitude of studies have been done relating telomere length to age, longevity, age-related disease, cancers, and stress. For decades, the gold standard of telomere length measurements was terminal restriction fragment analysis using Southern hybridization. However, recently an affordable, quick, and easy to perform method by Ca.......

Materials

Name	Company	Catalog Number	Comments
Name of the reagent	Company	Catalogue number
Quantitative Telomerase Detection	Allied Biotech	MT3012
Qiagen DNeasy Kit	Qiagen	69506
LightCycler 480 SYBR Green I Master, Ready-to-use hot start reaction mix for SYBR Green I-based real-time PCR using the LightCycler 480 Instrument	Roche Diagnostics	4707516