Optimization of Performance Parameters of the TAGGG Telomere Length Assay

Summary

Here, we describe in detail the protocol for quantifying telomere length using non-radioactive chemiluminescent detection, with a focus on the optimization of various performance parameters of the TAGGG telomere length assay kit, such as buffer quantities and probe concentrations.

Abstract

Telomeres are repetitive sequences which are present at chromosomal ends; their shortening is a characteristic feature of human somatic cells. Shortening occurs due to a problem with end replication and the absence of the telomerase enzyme, which is responsible for maintaining telomere length. Interestingly, telomeres also shorten in response to various internal physiological processes, like oxidative stress and inflammation, which may be impacted due to extracellular agents like pollutants, infectious agents, nutrients, or radiation. Thus, telomere length serves as an excellent biomarker of aging and various physiological health parameters. The TAGGG telomere length assay kit is used to quantify average telomere lengths using the telomere restriction fragment (TRF) assay and is highly reproducible. However, it is an expensive method, and because of this, it is not employed routinely for large sample numbers. Here, we describe a detailed protocol for an optimized and cost-effective measurement of telomere length using Southern blots or TRF analysis and non-radioactive chemiluminescence-based detection.

Introduction

Telomeres are the repetitive DNA sequences present at the end of chromosomes. They have tandem repeats of TTAGGG and maintain genome integrity by protecting the chromosome from both fraying and the end replication problem, which means that part of the 3' overhang is unable to be replicated by DNA polymerase^1,2. Short telomeres lead to chromosomal abnormalities in cells, due to which cells become permanently arrested in a stage called replicative senescence³. Short telomeres also cause a host of other problems, such as mitochondria dysfunction^4,

Protocol

NOTE: See the Table of Materials for details about all reagents used in the protocol below. Table 1 enlists lab-made reagents along with optimized volumes and Table 2 shows working concentrations of commercially available reagents.

1. Cell culture

1. Maintain cells whose telomere length is to be measured (used here were A2780 cells, which is an ovarian adenocarcinoma cell line) in Dulbecco's modified eagle medium (DMEM) complete medium supplemented with 10% fetal bovine serum (FBS), streptomycin, penicillin, and amphotericin B in a 6 cm Petri dish. Incubate at ....

Results

The extracted genomic DNA (gDNA), which was run on a 1% agarose gel, showed good integrity, as shown in Figure 1B, indicating that the sample could be used for further downstream processing of TRFs. The TRF assay was then carried out by the modifying the volumes of solutions required at each step (see Table 1 and Table 2). The TRF signal was clearly visible (Figure 3). Thus, by modifying the solution volumes and concentrations, .......

Discussion

We describe a detailed procedure for a non-radioactive, chemiluminescence-based method for telomere length measurement using Southern blotting. The protocol has been tested to allow the judicious use of several reagents with no compromise on the quality of results. The prehybridization and hybridization buffer can be reused up to five times. Enzyme concentration can vary between 10-20 U per 1.5-2 µg of genomic DNA without affecting the results. Several other kit components, such as the DIG-labeled molecular weight m.......

Materials

Name	Company	Catalog Number	Comments
Cell Line
A2780 (Ovarian adenocarcinoma cell line)	Received as a gift
Equipment
ChemiDoc XRS+ (for imaging and UV cross linking)	Biorad	Universal hood II (721BR14277)
Nanodrop (Epoch 2)	Biotek	EPOCH2
Software
TeloTool	Version 1.3
Materials
Acetic Acid	Molychem	64-19-7
Agarose	MP	180720
Amphotericin B	Gibco, ThermoFisher Scientific, USA	15240062
DMEM	HyClone, Cytiva, USA	SH30243.01
Ethylenediamine tetraacetic acid	Molychem	6381-92-6
HI FBS	Gibco, ThermoFisher Scientific, USA	10270106
HCl	Molychem	76-47-01-0
NaCl	Molychem	7647-14-5
NaOH	Molychem	1310-73-2
Nylon membrane	Sigma	11209299001
Penicillin	Gibco, ThermoFisher Scientific, USA	15240062
Sodium dodecyl sulfate	Affymetrix	151-21-3
Streptomycin	Gibco, ThermoFisher Scientific, USA	15240062
Tris	BIORAD	77-86-1
Tris HCl	Sigma Aldrich	1185-53-1
Whatman paper	GE healthcare lifesciences	1001-917
Reagents
1 kb ladder	NEB	N3232S
20x SSC	Invitrogen	15557-036
Anti DIG AP	Telo TAGGG Telomere Length Assay kit	12209136001
Blocking solution 10x	Telo TAGGG Telomere Length Assay kit	12209136001
Cutsmart Buffer	NEB	B6004
Detection buffer 10x	Telo TAGGG Telomere Length Assay kit	12209136001
Dig easy hyb	Telo TAGGG Telomere Length Assay kit	12209136001
Digestion Buffer	Telo TAGGG Telomere Length Assay kit	12209136001
Hinf 1	Telo TAGGG Telomere Length Assay kit	12209136001
Hinf 1 (alternative to kit)	NEB	R0155T
Loading Dye	BIOLABS	N3231S
Maleic acid buffer 10x	Telo TAGGG Telomere Length Assay kit	12209136001
Molecular marker	Telo TAGGG Telomere Length Assay kit	12209136001
Probe	Telo TAGGG Telomere Length Assay kit	12209136001
Rsa 1	Telo TAGGG Telomere Length Assay kit	12209136001
Rsa 1 (alternative to kit)	NEB	R0167L
Substrate	Telo TAGGG Telomere Length Assay kit	12209136001
Wash buffer	Telo TAGGG Telomere Length Assay kit	12209136001