Here we present a protocol for the measurement of relative telomere length (TL) using the monochrome multiplex quantitative polymerase chain reaction (MMqPCR) assay. The MMqPCR assay is a repeatable, efficient, and cost-effective method for measuring TL from human DNA in population-based studies.
Telomeres are ribonucleoprotein structures at the end of all eukaryotic chromosomes that protect DNA from damage and preserve chromosome stability. Telomere length (TL) has been associated with various exposures, biological processes, and health outcomes. This article describes the monochrome multiplex quantitative polymerase chain reaction (MMqPCR) assay protocol routinely conducted in our laboratory for measuring relative mean TL from human DNA. There are several different PCR-based TL measurement methods, but the specific protocol for the MMqPCR method presented in this publication is repeatable, efficient, cost-effective, and suitable for population-based studies. This detailed protocol outlines all information necessary for investigators to establish this assay in their laboratory. In addition, this protocol provides specific steps to increase the reproducibility of TL measurement by this assay, defined by the intraclass correlation coefficient (ICC) across repeated measurements of the same sample. The ICC is a critical factor in evaluating expected power for a specific study population; as such, reporting cohort-specific ICCs for any TL assay is a necessary step to enhance the overall rigor of population-based studies of TL. Example results utilizing DNA samples extracted from peripheral blood mononuclear cells demonstrate the feasibility of generating highly repeatable TL data using this MMqPCR protocol.
Telomeres are protective complexes found at the end of all eukaryotic chromosomes, composed of highly conserved, repetitive DNA sequences and associated proteins. The telomere protects the integrity of DNA, preserving chromosome stability. Progressive shortening of telomeres occurs in dividing cells as a result of incomplete lagging-strand DNA synthesis, DNA damage, and other factors1,2. Increased evidence supporting telomere length (TL) as a biomarker of aging and age-related diseases across the human lifespan has been accompanied by an increase in the types of TL measurement assays utilized to evaluate the role of TL in studies of human exposure, disease, and health3,4,5. Meta-analyses have reported associations of TL with overall mortality, environmental exposures, and health outcomes, including cancer, cardiovascular disease, and diabetes6,7,8,9,10,11,12,13. These meta-associations derive from studies utilizing one of over two dozen different TL measurement methodologies where the strengths of associations tend to vary between different methodologies14,15,16. Selecting the optimal TL measurement method for a research study is a crucial step to ensuring accurate results, as each method possesses its own advantages and disadvantages5,17.
Due to the relatively low cost of reagents, rapid assay turnaround, scalability, and lower initial DNA requirement, PCR-based TL measurement techniques are often preferentially utilized when conducting studies with large sample populations, studies with limited access to samples with high DNA concentrations, or studies prioritizing high throughput. The first PCR-based method of TL measurement, the singleplex quantitative polymerase chain reaction (qPCR) originally developed by Richard Cawthon, utilizes the ratio of the fluorescence signals of telomere (T) and single copy gene (S) amplification, run on separate PCR plates18. In this approach, primers complementary to the repeated telomere DNA sequences (T) are used to amplify total telomeric DNA content in the sample and quantified by detection of the fluorescent reporter SYBR green. Similarly, primers complementary to an intergenic region of a conserved single copy (S) gene are used to quantify genome copy number. These two estimates are quantified relative to a genomic DNA standard curve utilized across all assays in a project to control plate-to-plate variation. Dividing total telomeric DNA (T) by single genome copy number (S) produces the T/S ratio, a unitless, relative measurement representing average telomeric content per cell for an individual DNA sample18,19. Thus, the T/S ratio is not a specific measurement of functional length; however, consistent with literature norms, we utilize the term average TL per sample throughout this protocol.
This method was advanced in 2009 when Richard Cawthon described the monochrome multiplex qPCR (MMqPCR) assay as an approach to potentially reduce the variability in the T/S ratio relative to the original singleplex qPCR method19. The MMqPCR assay possesses the benefits of the qPCR assay with the additional advantage of measuring T and S signals within the same reaction well using one reporting fluorophore, thereby decreasing error relative to singleplex qPCR and resulting in higher precision and reproducibility19. Furthermore, this multiplex method potentially lowers costs and enhances throughput since half as many reactions are required compared to the singleplex assay19.
Given the advantages of MMqPCR TL measurement, this method is well-suited for population-based studies of TL associations with exposures, health outcomes, and biological processes. However, initiating the method can be challenging. To address these challenges, we describe, in detail, the MMqPCR TL measurement protocol utilized in our laboratory, highlighting key steps implemented to increase assay precision, decrease risk of contamination, and enhance repeatability.
Furthermore, this protocol outlines steps for cleaning data and calculating the intraclass correlation coefficient (ICC), an important statistical measure of the reproducibility of TL measurements2. With our representative results, we demonstrate the capability of generating high ICCs using this protocol. Additionally, we identify quality control (QC) and troubleshooting steps expected to decrease the variation in the TL measurements and increase the resulting ICC. Due to this method's high repeatability, efficiency, and cost-effectiveness, the MMqPCR TL measurement is ideal for epidemiological TL research. Figure 1 provides a visual overview of the MMqPCR method as described in this protocol.
Figure 1: Method overview. A broad overview of the monochrome multiplex quantitative polymerase chain reaction method for measuring telomere length. Please click here to view a larger version of this figure.
The method most commonly used TL measurement in larger population-based studies, prior to 2002, was the Southern blot analysis of terminal restriction fragment lengths (TRF)24,25. TRF, despite providing excellent precision and reproducibility in specialized laboratories, is limited in applicability due to the amount and quality of required DNA and limited throughput, thereby providing the backdrop for the increased utilization of qPCR-based TL assays, and subsequently the MMqPCR assay. The MMqPCR TL method provides repeatable TL measurement when set up, optimized, and maintained with careful attention to each QC criteria. The calculation and reporting of the specific ICC for each cohort analyzed is required to ensure assay reliability. While subject to DNA quality and technical expertise, the MMqPCR method is well-suited for large population-based studies investigating TL because it requires small amounts of DNA, is more reliable than singleplex PCR, and is more efficient in reagent costs and technician time than other methods. The ability to generate high ICCs provides additional data in support of the use of MMqPCR for large population-based studies of TL. MMqPCR TL measurement can be applied to a broad range of studies seeking to define the role of TL as a biomarker of all-cause mortality, aging, life-time stress, environmental exposures, and physical health outcomes such as cardiovascular disease and cancer4,7,8,10,11,12,13,26,27,28,29,30,31.
One limitation of the MMqPCR method is that it reports TL as a T/S ratio, a relative estimate of length that varies depending upon the selection of single-copy gene, master mix composition, and PCR cycling parameters32. The T/S ratio is unitless. Thus, without combining with other TL measurement methodologies, this method is incapable of reporting estimates in base pair values18,33,34. As a result, the T/S ratio must be transformed to a Z-score to have relevance across studies35. Substantial caution should be taken when doing this across laboratories, methods, and assays. Further, this method, as with gel-based hybridization assays, can include quantification of interstitial telomeres. However, these sequences comprise a very small proportion of total telomere DNA content per genome. In addition, interstitial telomeric sequences are more likely to contain mismatched base pair sequences which deviate from canonical telomere repeats, decreasing the likelihood of primer binding and amplification. Additionally, while the minimal amount of DNA needed for the MMqPCR assay is advantageous, it is important to note that qPCR-based measurements of TL are influenced by pre-analytical factors impacting DNA quality and integrity, including sample storage conditions, DNA extraction methodology, and biological tissue36,37. It has been shown that analytical control for these factors can enhance the external validity of TL measurements generated using qPCR38. Even so, the impact of differences in DNA quality on TL generated by MMqPCR assay specifically needs to be systematically evaluated, as no current data-informed guidance exists for determining if a sample is of sufficient quality to generate an accurate estimate of TL using this approach. Despite these limitations, the applications of this assay for studies on population level health outcomes are considerable.
When utilizing the MMqPCR assay, continual assessment of precision and throughput is needed. As currently designed, technicians run triplicates of samples simultaneously on duplicate plates using two thermocyclers. In the absence of multiple thermocyclers, we recommend retaining duplicate-triplicate measurements and running plates sequentially for enhanced precision even at the cost of diminished throughput. Any decision to prioritize throughput over precision, for example by employing single triplicate measurements, should be accompanied with thorough testing and evaluation of the resulting ICCs before proceeding with analysis of analytical samples. When making decisions regarding throughput and precision, one must take into consideration the sample size and the quality of the sample DNA. A smaller sample size or poor-quality DNA samples necessitates prioritizing higher precision23. This is of even greater importance when working with samples from related groups (e.g., family members, subjects with multiple timepoints). In these instances, careful planning, for example assigning related samples to the same plate prior to starting the experiment, is one way to prevent loss of statistical power through inadvertent group by plate confounding.
For this assay, a spectrophotometer was used to assess DNA sample quality: samples within the range of 1.6-2.0 for 260/280 ratios and 2.0-2.2 for 260/230 ratios were considered acceptable. This quality assessment and the accurate assessment of double-stranded DNA via fluorometer are critical steps in this protocol for obtaining repeatable TL data. Other, more descriptive measures of DNA integrity, such as fragment size and/or summary measures of DNA quality determined via agarose gel (e.g., DNA integrity number) may also be utilized in determining sample quality38. We also recommend that the dilution of samples only occur at the time of sample preparation for running the MMqPCR assay. This ensures that DNA aliquots undergo the least amount of manipulation after extraction as possible, decreasing the variability in pre-assay handling. Should DNA samples need to be transported, they should be shipped on dry ice at the highest possible concentration to mitigate the degradation that occurs in DNA samples at lower concentrations39,40. Due to the degradation of DNA that occurs with freeze-thaw cycles, the number of freeze-thaws to stock DNA should be minimized41. Aliquots of the pooled control DNA should be created before running the cohort and aliquots of individual analytical DNA samples should be created prior to running the assay.
Key metrics of assay performance and QC include NTC signal, interplate and intraplate CVs, and standard curve R2. Failure to meet QC criteria can be mitigated in several ways. Changing PCR grade H2O stocks regularly and aliquoting PCR grade H2O sub-stocks for each pair of plates run will minimize sources of contamination as well as NTC amplification. Additional steps to abate contamination include the following: designating a specific PCR hood dedicated to only MMqPCR assay; wiping down the PCR hood and equipment with a DNA decontaminating solution; irradiating the room with ultra-violet light; and practicing sterile technique when in the PCR hood. To enhance assay repeatability and decrease CVs, it is recommended to vortex samples, dilutions, and the PCR strips vigorously at their respective vortex steps and resuspend thoroughly when pipetting DNA samples. A below threshold standard R2 curve (<0.995) is most likely attributable to pipetting errors during plate loading. To avoid this, pay careful attention to precise pipetting and calibrate pipets annually, vigorously mix standard PCR strips before loading, and carefully organize supplies to promote an efficient workflow. If using two machines and one plate is observed to output consistently higher CVs, the machine should be serviced as a potential way to ameliorate the problem. QC plates from the manufacturer should be run on the plates regularly to assess thermocycler's performance.
If problems persist even after application of the steps recommended above, the following steps can be used to troubleshoot the protocol. Keeping a record of when all reagents were aliquoted and any pertinent expiration dates can help streamline the troubleshooting process when difficulties inevitably arise. An important part of any troubleshooting process is to only adjust one reagent at a time to determine the specific cause of plates not passing the QC criteria, starting with the least expensive reagent in question. For example, if both the telomere and single copy gene have low efficiencies, shared reagents such as the DTT, dNTPs, or SYBR aliquot are more likely the cause than amplicon specific primers. At the listed price, new aliquots should be tested in order of DTT, then dNTPs, and then finally, if the issue still persists, new SYBR aliquots. Conversely, if only one of amplicons (telomere or single copy gene) has a low efficiency, the cause of difficulty is more likely one of the primers. Interpretation of the melt curve peaks presented in Figure 8 can serve as a source of key information for troubleshooting. Visualization of the two melt curve peaks can be used to identify potential issues with a particular sample, as an individual problem sample will stand out from the general trend of peaks exhibited by standards or remaining analytical samples. The melt curve can also be used to diagnose problems with a particular primer if the peaks for a given amplicon are systematically less sharp than the other.
This manuscript details how to successfully set up the MMqPCR assay for measuring TL with broad applicability to public health research and introduces key recommendations for QC and troubleshooting with the goal of increasing accessibility and reliability of this efficient and cost-effective method.
The authors have nothing to disclose.
The authors would like to acknowledge the Telomere Research Network Advisory Committee and the National Institute on Aging / National Institute of Environmental Health Sciences funding (U24 AG066528 and U24 AG066528-S1) that have made this work possible.
0.5mL Tubes | USA Scientific | 1605-0099 | Seal-Rite 0.5mL Microcentrifuge Tubes, Sterile Storage Temperature and Conditions: Room temperature |
1.5mL Tubes | USA Scientific | 1615-5599 | Seal-Rite 1.5mL Microcentrifuge Tubes, Sterile Storage Temperature and Conditions: Room temperature |
100mM DTT | In House | Not Applicable | Made with stock DTT, diluted sodium acetate, and PCR Grade H2O Storage Temperature and Conditions: minus 20 °C |
15mL Tubes | Thermo Fisher | 14-959-53A | Corning 352196 Falcon 15mL Conical Centrifuge Tubes Storage Temperature and Conditions: Room temperature |
1M MgCl2 | Thermo Fisher | 50152107 | Biotang Inc 1M MgCl2 1M Magnesium Chloride Solution, Prepared in 18.2 Megohms Water and Filtered through 0.22 Micron Filter Storage Temperature and Conditions: 4 °C |
1x Gold Buffer | In House | Not Applicable | 10X Gold Buffer diluted with PCR Grade H2O Storage Temperature and Conditions: minus 20 °C |
25mM dNTPs | New England BioLabs | N0446S | Deoxynucleotide Solution Set Storage Temperature and Conditions: minus 20 °C |
5mL Tubes | Thermo Fisher | 3391276 | Argos Technologies Microcentrifuge Tubes – 5mL Storage Temperature and Conditions: Room temperature |
96 Well Plate | Bio-Rad | HSP9601 | Hard-Shell 96-Well PCR Plates, Low Profile, Thin Wall, Skirted, White / Clear Storage Temperature and Conditions: Room temperature |
Aluminum Foil | Office Depot | 3489072 | Reynolds Wrap Stanard Aluminum Foil Roll, 12" x 75', Silver Storage Temperature and Conditions: Room temperature |
AmpliTaq Gold Kit – Polymerase and Buffer | Thermo Fisher | 4311806 | AmpliTaq Gold DNA Polymerase with Gold Buffer and MgCl2 (MgCl2 in this kit is not used), 10X Gold Buffer, 2.5U AmpliTaq Gold Polymerase Storage Temperature and Conditions: minus 20 °C |
Betaine | Thermo Fisher | AAJ77507AB | Betaine, 5M Solution, Molecular Biology Grade, Ultrapure, 10mL Storage Temperature and Conditions: minus 20 °C |
Big Tube Rack | Thermo Fisher | 344817 | Fisherbrand 4-Way Tube Rack Storage Temperature and Conditions: Room temperature |
CFX Maestro Software | Bio-Rad | 12004110 | Software for real-time PCR plate setup, data collection, statistics, and graphiing of results Storage Temperature and Conditions: Room temperature |
CFX96 Optical Reaction Module for Real-Time PCR Systems with Starter Package | Bio-Rad | 1845096 | 96-well optical module for real-time PCR Storage Temperature and Conditions: Room temperature |
DTT | Fisher Scientific | AAJ1539706 | Dithiothreitol, >99.5+ Molecular Biology Grade, 5 g Storage Temperature and Conditions: minus 20 °C |
ELIMINase | Fisher Scientific | 04-355-32 | ELIMINase Laboratory Decontaminant Storage Temperature and Conditions: Room temperature |
HEPA Filter | USA Scientific | Replacement Filters | High-Efficiency Particulate Air Filter for AirClean Workstations Storage Temperature and Conditions: Room temperature |
Kimwipes | Thermo Fisher | 06666A | Kimberly-Clark Professional Kimtech Science Kimwipes Delicate Task Wipers, 1-Ply Storage Temperature and Conditions: Room temperature |
Loading Trough | Thermo Fisher | 14387069 | Thermo Scientific Matrix Reagent Reservoirs Storage Temperature and Conditions: Room temperature |
Microsoft Excel | Microsoft | Not Applicable | Microsoft 365 package, Excel software application Storage Temperature and Conditions: Room temperature |
Mini Centrifuge | Genesee Scientific | 31-500B | Poseidon 31-500B Mini Centrifuge, Blue Lid Storage Temperature and Conditions: Room temperature |
PCR Grade H2O | Thermo Fisher | AM9937 | Nuclease-Free Water (not DEPC-Treated) Storage Temperature and Conditions: Room temperature |
PCR Hood | USA Scientific | 4263-2588 | Nucleic Acid Workstation with HEPA Filtration, AirClean Systems Combination PCR Workstation Storage Temperature and Conditions: Room temperature |
PCR Strips | Thermo Fisher | AB0776 | Low Profile Tubes and Flat Caps, Strips of 8 Storage Temperature and Conditions: Room temperature |
PCR Tube Rack | Thermo Fisher | 344820 | Fisherbrand 96-Well PCR Tube Rack Storage Temperature and Conditions: Room temperature |
Pipette Tips (Multichannel) | Ranin | 17005860 | Pipette Tips SR LTS 20µL F 960A/5, 20µL Maximum Storage Temperature and Conditions: Room temperature |
Pipette Tips (Single Channel) | USA Scientific | 1181-3850 | 10µL Graduated TipOne RPT Filter Tips Storage Temperature and Conditions: Room temperature |
Pipette Tips (Single Channel) | USA Scientific | 1180-1850 | 20µL Beveled TipOne RPT Filter Tips Storage Temperature and Conditions: Room temperature |
Pipette Tips (Single Channel) | USA Scientific | 1111-0880 | 200µL Natural TipOne Pipette Tips in Racks Storage Temperature and Conditions: Room temperature |
Pipette Tips (Single Channel) | USA Scientific | 1111-2890 | 1000µL Natural Graduated TipOne Pipette Tips in Racks Storage Temperature and Conditions: Room temperature |
Pipettors (Multichannel) | Ranin | 17013802 | Pipet-Lite Multi Pipette L8-10XLS, 0.5 to 10µL Storage Temperature and Conditions: Room temperature |
Pipettors (Multichannel) | Ranin | 17013803 | Pipet-Lite Multi Pipette L8-20LS+, 2 to 20µL Storage Temperature and Conditions: Room temperature |
Pipettors (Single Channel) | Thermo Fisher | F144802G | Gilson Pipetman Classic Pipets, 1 to 10µL Storage Temperature and Conditions: Room temperature |
Pipettors (Single Channel) | Thermo Fisher | F123600 | Gilson Pipetman Classic Pipets, 2 to 20µL Storage Temperature and Conditions: Room temperature |
Pipettors (Single Channel) | Thermo Fisher | F123601 | Gilson Pipetman Classic Pipets, 20 to 200µL Storage Temperature and Conditions: Room temperature |
Pipettors (Single Channel) | Thermo Fisher | F123602 | Gilson Pipetman Classic Pipets, 200 to 1000µL Storage Temperature and Conditions: Room temperature |
Plate Sealing Film | Bio-Rad | MSB1001 | Microseal “B” PCR Plate Sealing Film, Adhesive, Optical Storage Temperature and Conditions: Room temperature |
Plate Spinner | Thermo Fisher | 14-100-141 | Fisherbrand Mini Plate Spinner Centrifuge, 230 V Storage Temperature and Conditions: Room temperature |
Pre-Hood Filter | USA Scientific | 4235-3724 | Prefilter for AirClean Systems Workstations Storage Temperature and Conditions: Room temperature |
R Software | The R Project for Statistical Computing | Not Applicable | R version 4.2.2 Storage Temperature and Conditions: Room temperature |
Scale | Thermo Fisher | 01-922-329 | OHAUS 30430060 PR Series Analytical Balance, 62g Capacity Storage Temperature and Conditions: Room temperature |
Scissors | Office Depot | 458612 | Office Depot Brand Scissors, 8”, Straight, Black, Pack of 2 Storage Temperature and Conditions: Room temperature |
Sharpies | Sharpie | 2151734 | Brush Twin Permanent Markers, Black Storage Temperature and Conditions: Room temperature |
Single Copy Gene Forward Primer | Integrated DNA Technologies | Custom | See separate table Storage Temperature and Conditions: minus 20 °C when rehydrated |
Single Copy Gene Reverse Primer | Integrated DNA Technologies | Custom | See separate table Storage Temperature and Conditions: minus 20 °C when rehydrated |
Small Tube Rack | Thermo Fisher | 21-402-17 | Thermo Fisher 8601 Reversible Microtube Racks with Lid Storage Temperature and Conditions: Room temperature |
Sodium Acetate | Thermo Fisher | J63560.EQE | 3M NaOAc pH 5.2 Storage Temperature and Conditions: Room temperature |
Stainless Steel Spatula | Thermo Fisher | 3990240 | Bel-Art SP Scienceware Stainless-Steel Sampling Spoon and Spatula Storage Temperature and Conditions: Room temperature |
SYBR Green | Thermo Fisher | S7563 | SYBR Green I Nucleic Acid Gel Stain – 10,000X Concentrate in DMSO Storage Temperature and Conditions: minus 20 °C when rehydrated |
Syringe Filters | Fisher Scientific | 09-927-55A | GD/X 25 mm Sterile Syringe Filter, cellulose acetate filtration medium, 0.2 μm Storage Temperature and Conditions: Room temperature |
Syringes | Thermo Fisher | 148232A | BD Luer-Lok Disposable Syringes without Needles, 10mL Storage Temperature and Conditions: Room temperature |
TE Buffer | Fisher Scientific | BP2474100 | TE Buffer, Tris-EDTA, 1X Solution, pH 7.6, Molecular Biology, Fisher BioReagents Storage Temperature and Conditions: Room temperature |
Telomere Forward Primer | Integrated DNA Technologies | Custom | See separate table Storage Temperature and Conditions: minus 20 °C when rehydrated |
Telomere Reverse Primer | Integrated DNA Technologies | Custom | See separate table Storage Temperature and Conditions: minus 20 °C when rehydrated |
UV Light | USA Scientific | 4288-2540 | UV Light Bulb for Workstations Storage Temperature and Conditions: Room temperature |
Vortex | Thermo Fisher | 14-955-151 | Fisherbrand Mini Vortex Mixer, 115 V, 50/60 Hz Storage Temperature and Conditions: Room temperature |
Weigh Boat | Thermo Fisher | 01-549-752 | Fisherbrand Sterile Hexagonal Weighing Boat, 10mL Storage Temperature and Conditions: Room temperature |
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