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^4,5 and cell dysfunction.

DNA telomeric repeats are lost as and when the cell divides, with an average loss of 25 to 200 bp per year⁶, resulting in cellular senescence after a certain number of divisions⁶. Aging is associated with a higher frequency of comorbidities, which is marked by a shortening in telomere length⁷. Telomere restriction fragment (TRF) analysis, as described by Mender, is a very expensive method⁸. Because of this, it is not implemented while quantifying telomere length in most studies.

Presently, the majority of epidemiological studies employ quantitative polymerase chain reaction (qPCR)-based measurements of telomere length. However, the qPCR-based method is a relative measurement method, as it measures the ratio between telomeres and single-copy gene amplification products, and not absolute telomere length. Telomere length measurement using the TRF protocol is the gold standard method, as it can measure telomere length distribution in the sample and measurements can be expressed in absolute values in kilobases (kb). However, its use is limited because it is cumbersome, labor-intensive, and costly. Here, we present an optimized protocol for telomere length measurement using chemiluminescence-based TRFs.

TRF analysis includes seven major steps: 1) culturing of cells for genomic DNA extraction, 2) genomic DNA extraction using the phenol:chloroform:isoamylalcohol (P:C:I) method, 3) restriction digestion of genomic DNA, 4) agarose gel electrophoresis, 5) Southern blotting of the restriction digestion DNA fragment, 6) hybridization and detection via chemiluminescence-the immobilized telomere probe is visualized by a highly sensitive chemiluminescent substrate for alkaline phosphatase, disodium 2-chloro-5-(4-methoxyspiro[1,2-dioxetane-3,2′-(5-chlorotricyclo[3.3.1.13.7]decan])-4-yl]-1-phenyl phosphate (CDP-Star)-and 7) analysis for obtaining mean telomere length and range information from these telomeric smears.

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 37 °C in a humidified and controlled environment containing 5% carbon dioxide until the cells are 80%-100% confluent.
2. Remove the media and wash with 5 mL of 1x phosphate-buffered saline (PBS).
3. Treat the cells with 1 mL of trypsin-ethylenediaminetetraacetic acid (EDTA) for detaching the cells by incubating at 37 °C for 3-5 min.
4. Add 2 mL of DMEM complete media to inactivate the trypsin and collect the cells in a centrifugation tube.
5. Pellet the cells at 2,348 × g for 5 min.
6. Wash the pellet with 1x PBS and centrifuge at 2,348 × g for 5 min.
7. Store the pellet at -80 °C until further use.
   ​NOTE: The cell count can vary depending on the cell line. We obtained approximately 2.5 × 10⁶ cells in the case of the A2780 cell line, which is used in further steps.

2. Genomic DNA isolation

1. Add 500 µL of lysis buffer (10 mM tris-Cl, pH 8.0; 25 mM EDTA, pH 8.0; 100 mM NaCl; 0.5% w/v sodium dodecyl sulphate) to the cell pellet and gently mix using a cut tip (with an opening diameter of a minimum 2 mm). Add 20 µg/mL freshly prepared RNase A and mix gently by inversion.
2. Incubate at 37 °C for 30 min and occasionally invert the tube during incubation.
3. Add proteinase K to a final concentration of 100 µg/mL and mix gently by inversion 10 times. Incubate at 55 °C for 2 h. Invert the tube at regular intervals (every 10 min) during incubation.
4. Add 500 µL of phenol:chloroform:isoamyl alcohol reagent (25:24:1) and mix gently by inversion 20 times. Centrifuge at 9,391 × g for 15 min at room temperature (around 25 °C).
   NOTE: Figure 1A shows the three layers obtained after centrifugation.
5. Remove the viscous upper aqueous layer out of the three layers visible, place into a fresh tube using a cut tip, and add an equal amount of chloroform. Mix by gentle inversion 20 times.
6. Centrifuge the tubes at 9,391 × g for 15 min at room temperature.
7. Collect the aqueous layer and add an appropriate amount of 5 M NaCl so that the final concentration of NaCl is 0.2 M.
8. Add two volumes of 100% ethanol. Mix by gentle inversion 20-25 times. Centrifuge at 15,871 × g for 5 min at room temperature.
9. Remove the supernatant and add 500 µL of 70% ethanol to wash the pellet. Centrifuge at 15,871 × g for 5 min at room temperature.
10. Remove the supernatant, air-dry the pellet for a few minutes, and add 50 µL of sterile nuclease-free water. Allow the DNA to rehydrate for 1-2 days at room temperature. Mix by pipetting, using the cut tip.
11. Measure the concentration of DNA using ultraviolet (UV)-spectrophotometry. Redilute the samples, if necessary, using sterile nuclease-free water to get a minimum concentration of 300-500 ng/µL.
12. Check for the integrity of the DNA by running it on a 1% agarose gel (Figure 1B).
13. Store the diluted DNA at -20 °C until further use.

3. Digestion of genomic DNA

1. Prepare an enzyme mixture of Rsa1 (20 U) and Hinf1 (20 U) and add 2 µL of restriction digestion buffer per reaction.
2. Take an appropriate volume of genomic DNA such that the total amount of DNA is 1.5 µg. Make up the volume with sterile nuclease-free water, so that the total volume after the enzyme addition is 20 µL.
3. Mix well by tapping, followed by a pulse spin.
4. Incubate the mixture at 37 °C for 2 h.

4. Agarose gel electrophoresis

1. Prepare a 10 cm × 15 cm 0.8% agarose gel in 1x tris acetate EDTA (TAE) buffer using high-grade agarose.
2. Add 5 µL of loading dye to each digested genomic DNA sample, thus making the final volume 25 µL, and load the samples onto the gel.
3. Prepare a molecular marker mix by using 1 µL of molecular marker (ladder), 3 µL of sterile nuclease-free water, and 1 µL of 5x loading dye.
4. Load 5 µL of molecular marker mix on both sides of the genomic DNA digested samples if there are a higher number of samples (~10), or only on one side if there are less than or equal to five samples.
5. Run the gel at 5 V/cm for 6 h. Once the run is complete, make a notch on one corner of the gel to mark the loading order.
6. Submerge the gel in 0.25 M HCl for 10 min at room temperature with gentle agitation.
7. Rinse the gel twice with distilled water.
8. Denature the DNA by submerging the gel in a solution of 0.5 M NaOH and 1.5 M NaCl twice for 15 min each at room temperature with gentle agitation.
9. Rinse the gel twice with distilled water.
10. Neutralize the DNA by submerging the gel in a solution of 0.5 M tris-HCl and 3 M NaCl (pH 7.5) twice for 15 min at room temperature with gentle agitation.

5. Southern blotting

1. Cut a nylon membrane of 10 cm × 12 cm in size and make a notch in the same position as the gel.
2. Activate the membrane by dipping in distilled water followed by 20x sodium saline citrate (SSC) buffer (see Table 1).
3. Set up the transfer.
   1. Take a clean glass tray and place another tray in it in an inverted position. Create a wick using regular filter paper such that the ends are touching the base of the outer tray.
   2. Place the gel on top of the filter paper. Gently place the activated nylon membrane over the gel, with the notches overlapping, and remove any air bubbles by rolling over a glass rod.
   3. Place a 2 cm heap of 9.5 cm × 11.5 cm cellulose filter paper, followed by a 6 cm heap of regular filter paper of the same dimensions. Place a weight atop this setup such that there is equal weight distribution below. Fill the outer tray with 20x SSC buffer (see Figure 2).
   4. Leave the setup for overnight transfer.
4. After the Southern transfer, fix the transferred DNA onto the membrane by UV crosslinking on a UV transilluminator or UV crosslinker. Use a 302 nm 8 W lamp for 5 min, or a 254 nm 8 W lamp for 2 min, which corresponds to approximately 120 mJ. Ensure that the membrane side on which the DNA is transferred faces the lamp.
5. Wash the membrane twice with 25 mL of 2x SSC buffer.
6. Pause the protocol, if required, by completely drying the blot and storing it at 2-8 °C after loosely wrapping in foil, till further processing.

6. Hybridization and chemiluminescence detection

1. Prewarm the prehybridization buffer to 42 °C.
   NOTE: The following steps include volumes of solutions/buffers to be used per 100 cm² of blot.
2. Incubate the membrane in 10 mL of prehybridization buffer for 1 h at 42 °C with gentle agitation for prehybridization.
3. Add 0.5 µL of telomere probe per 5 mL of prewarmed prehybridization buffer to make the hybridization solution.
4. Incubate the blot in 10 mL of hybridization solution at 42 °C for 3 h with gentle agitation.
5. Wash the blot with stringent buffer 1 (Table 1) twice for 10 min (25 mL each) at room temperature with gentle agitation.
6. Prewarm stringent buffer 2 (Table 1) for 30 min at 50 °C.
7. Wash the blot with stringent buffer 2 twice for 15 min (25 mL each) at 50 °C with gentle agitation.
8. Rinse with 15 mL of wash buffer for 5 min with gentle agitation at RT.
9. Incubate the membrane in 10 mL of freshly prepared 1x blocking solution for 30 min at room temperature with gentle agitation.
10. Incubate the membrane in 10 mL of anti-digioxigenin conjugated with alkaline phosphatase working solution (see Table 2) for 30 min at RT with gentle agitation.
11. Wash the blot twice with wash buffer at room temperature for 15 min with gentle agitation.
12. Incubate in 10 mL of detection buffer for 5 min at RT with gentle agitation.
13. Remove the excess detection buffer and keep the membrane with the DNA facing upward on a hybridization bag or acetate sheet. Add ~1-1.5 mL of substrate solution dropwise onto the membrane, immediately place another sheet on it, and incubate for 5 min at RT.
14. Squeeze out the excess substrate solution for imaging.
15. Image the blot for approximately 20 min in a gel documentation imaging system, collecting multiple images at different time points. Select unsaturated images for further analysis.
    ​NOTE: In case the signal is weak, imaging can be carried out for a longer period of time.

7. Analysis

1. After obtaining the image files, export them for publication in .tif format at the highest resolution possible (600 dpi in this case).
2. Install Telotool software. This requires a specific version (R2012a) of MATLAB (freely available) to run.
3. Open the software and click on the Load Image File button in the top right.
4. After the image has been loaded, click Invert Image, so that the image background is black and the smears/bands are white.
5. Crop the image with the top corners starting from the wells. After the crop selection has been made, right-click inside it and select Crop Image.
6. After the image has been cropped, click on Calculate Lanes and allow lane detection to occur automatically.
7. If the lanes have not been detected properly, for example, if certain lanes have not been detected at all, or if extra or partial lanes have been detected, perform lane adjustment as described below.
   1. Click on Adjust Lanes and in the pop-up window that opens, add and/or adjust lanes as needed, and click on Apply and Close.
8. After adjusting the lanes, make sure that a red dot is seen on each of the lanes and adjust the ladders using the Ladder Fit button, making sure that the number of peaks in both the ladder lanes are in the same position. Remove any extraneous peaks using the Delete Extremum button.
9. After the ladders have been adjusted, select Lane Profiles, click on Ladder Fit, and select Polynomial Fit.
10. Click on Trendline, as recommended by the software, and then select Corrected in the next option.
11. Click on Get Results to display the results as a table, in which the Mean TRF row is the telomere length measurement of the respective lane samples.
12. Click on Save all to store the results in the form of a spreadsheet.

Representative 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, more samples could be processed without any negative effect on the results, and the telomere length could be determined successfully using freely available software such as Telotool¹⁰.

Figure 1: Isolation and quality check of genomic DNA. (A) Three distinct separation phases obtained upon centrifugation after adding phenol:chloroform:isoamyl alcohol. The upper aqueous layer contains the gDNA, the interphase contains the proteins, and the lower, organic phase contains the degraded RNA, cell debris, and lipids. (B) An image of a 1% agarose gel showing intact undigested gDNA. Lane 1 shows a 1 kb ladder and lane 2 shows undigested gDNA of the cancer cell line A2780. Abbreviation: gDNA = genomic DNA. Please click here to view a larger version of this figure.

Figure 2: Illustration of Southern blotting transfer setup. Representative diagram showing the system assembly for the transfer of telomere repeat smears from the gel to the nylon membrane by capillary action. Please click here to view a larger version of this figure.

Figure 3: Chemiluminescence detection of TRFs post-Southern blotting and hybridization. Blot showing a range of telomeric repeats as a smear in lane 2. Lane 1 shows a molecular marker, with the molecular weights (kb) of bands indicated on left side. Please click here to view a larger version of this figure.

Table 1: List of reagents used in this protocol. The table contains reagents prepared in the lab along with their storage details, the recommended usage of the TAGGG telomere length assay kit reagents, and their modified usage volumes, as per the optimization in this protocol. Please click here to download this Table.

Table 2: List of commercially available reagents with modified usage instructions. The table contains commercially available reagents along with different dilutions, their storage details, and their recommended usage by the TAGGG telomere length assay kit and modified usage volumes, as per the optimization in this protocol. Please click here to download this Table.

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 marker and hybridization probe, can be used at lower concentrations than recommended. The optimized volumes are indicated in Table 2. We have tested them at half the recommended volumes/concentrations and found optimal performance. Following these steps reduces the cost per sample tremendously, thus enabling researchers to measure telomere length using this method at a larger scale.

There are several published TRF protocols. We have optimized the commercially available kit protocol to make it cost-effective. This protocol is different from some of the published protocols as it does not use radioactivity^11,12. It is also relatively simple, as it uses the kit components rather than preparing in-house reagents, particularly the telomere probe¹³.

If the final image is patchy, then the membrane has partially dried during incubation. To resolve this, one should either increase the solution volume or agitate at a higher speed. If there are no smears visible on the blot, then there might have been a problem during the Southern transfer. In addition, there could have been a problem in the DNA quantity or quality.

There are some limitations to this method. First, genomic DNA extraction and quantification takes upward of 4 days based on the source of the DNA¹⁴. Higher molecular weight DNA takes a longer time to rehydrate and homogenize, making quantification and accurate pipetting difficult. Second, the TRF protocol is long (2 days minimum) and requires skilled lab workers to implement. It is also an expensive method due to the reagents and the quantities required. The TRF protocol also measures the average telomere length in each sample, as opposed to the shortest telomere length that TESLA measures or the telomere length per cell that flow cytometry-based methods provide¹⁵. Another limitation of TRF analysis using the enzymes used here is an overestimation of telomere length, as these enzymes do not have a restriction site in the sub-telomeric regions¹⁵.

However, despite the limitations, there are reasons why this method is still considered the gold standard of telomere length measurement. Telomere lengths are rendered accurately in absolute values-in kilobase pairs (kbp).

This method can be used to measure the average telomere length in a variety of cell types, from cell lines^16,17 to buffy coat pellets^18,19. This makes it a robust method to use in several types of research studies. It can also be used in large-scale epidemiological studies as well as in studies where cell-based therapeutics are being developed.

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