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.

Accurate Telomere Length Assessment in Human Population Studies

<ol>
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The authors have nothing to disclose.

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&#160;are influenced by pre-analytical factors impacting DNA quality and integrity, including sample storage conditions, DNA extraction methodology, and biological tissue36,37. It has&#160;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&#160;(&#60;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&#39;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&#160;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.

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&#160;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&#160;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&#39;s high repeatability, efficiency, and cost-effectiveness, the MMqPCR TL measurement is ideal for epidemiological TL research. Figure 1&#160;provides a visual overview of the MMqPCR method as described in this protocol.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66545/66545fig01v2.jpg" /> 
Figure 1: Method overview. A broad overview of the monochrome multiplex quantitative polymerase chain reaction method for measuring telomere length. <a href="https://www.jove.com/files/ftp_upload/66545/66545fig01largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.5mL Tubes</td>
			<td>USA Scientific</td>
			<td>1605-0099</td>
			<td>Seal-Rite 0.5mL Microcentrifuge Tubes, Sterile 
			Storage Temperature and Conditions: Room temperature</td>
		</tr>
		<tr>
			<td>1.5mL Tubes</td>
			<td>USA Scientific</td>
			<td>1615-5599</td>
			<td>Seal-Rite 1.5mL Microcentrifuge Tubes, Sterile 
			Storage Temperature and Conditions: Room temperature</td>
		</tr>
		<tr>
			<td>100mM DTT</td>
			<td>In House</td>
			<td>Not Applicable</td>
			<td>Made with stock DTT, diluted sodium acetate, and PCR Grade H2O 
			Storage Temperature and Conditions: minus 20 &#176;C&#160;</td>
		</tr>
		<tr>
			<td>15mL Tubes</td>
			<td>Thermo Fisher</td>
			<td>14-959-53A</td>
			<td>Corning 352196 Falcon 15mL Conical Centrifuge Tubes 
			Storage Temperature and Conditions: Room temperature</td>
		</tr>
		<tr>
			<td>1M MgCl2</td>
			<td>Thermo Fisher</td>
			<td>50152107</td>
			<td>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 &#176;C&#160;</td>
		</tr>
		<tr>
			<td>1x Gold Buffer</td>
			<td>In House</td>
			<td>Not Applicable</td>
			<td>10X Gold Buffer diluted with PCR Grade H2O 
			Storage Temperature and Conditions: minus 20 &#176;C&#160;</td>
		</tr>
		<tr>
			<td>25mM dNTPs</td>
			<td>New England BioLabs</td>
			<td>N0446S</td>
			<td>Deoxynucleotide Solution Set 
			Storage Temperature and Conditions: minus 20 &#176;C&#160;</td>
		</tr>
		<tr>
			<td>5mL Tubes</td>
			<td>Thermo Fisher</td>
			<td>3391276</td>
			<td>Argos Technologies Microcentrifuge Tubes &#8211; 5mL 
			Storage Temperature and Conditions: Room temperature</td>
		</tr>
		<tr>
			<td>96 Well Plate</td>
			<td>Bio-Rad</td>
			<td>HSP9601</td>
			<td>Hard-Shell 96-Well PCR Plates, Low Profile, Thin Wall, Skirted, White / Clear 
			Storage Temperature and Conditions: Room temperature</td>
		</tr>
		<tr>
			<td>Aluminum Foil</td>
			<td>Office Depot</td>
			<td>3489072</td>
			<td>Reynolds Wrap Stanard Aluminum Foil Roll, 12&#34; x 75&#39;, Silver 
			Storage Temperature and Conditions: Room temperature</td>
		</tr>
		<tr>
			<td>AmpliTaq Gold Kit &#8211; Polymerase and Buffer</td>
			<td>Thermo Fisher</td>
			<td>4311806</td>
			<td>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 &#176;C&#160;</td>
		</tr>
		<tr>
			<td>Betaine</td>
			<td>Thermo Fisher</td>
			<td>AAJ77507AB</td>
			<td>Betaine, 5M Solution, Molecular Biology Grade, Ultrapure, 10mL 
			Storage Temperature and Conditions: minus 20 &#176;C&#160;</td>
		</tr>
		<tr>
			<td>Big Tube Rack</td>
			<td>Thermo Fisher</td>
			<td>344817</td>
			<td>Fisherbrand 4-Way Tube Rack 
			Storage Temperature and Conditions: Room temperature</td>
		</tr>
		<tr>
			<td>CFX Maestro Software</td>
			<td>Bio-Rad</td>
			<td>12004110</td>
			<td>Software for real-time PCR plate setup, data collection, statistics, and graphiing of results 
			Storage Temperature and Conditions: Room temperature</td>
		</tr>
		<tr>
			<td>CFX96 Optical Reaction Module for Real-Time PCR Systems with Starter Package</td>
			<td>Bio-Rad</td>
			<td>1845096</td>
			<td>96-well optical module for real-time PCR 
			Storage Temperature and Conditions: Room temperature</td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Fisher Scientific</td>
			<td>AAJ1539706</td>
			<td>Dithiothreitol, &#62;99.5+ Molecular Biology Grade, 5 g 
			Storage Temperature and Conditions: minus 20 &#176;C&#160;</td>
		</tr>
		<tr>
			<td>ELIMINase</td>
			<td>Fisher Scientific</td>
			<td>04-355-32</td>
			<td>ELIMINase Laboratory Decontaminant 
			Storage Temperature and Conditions: Room temperature</td>
		</tr>
		<tr>
			<td>HEPA Filter</td>
			<td>USA Scientific</td>
			<td>Replacement Filters</td>
			<td>High-Efficiency Particulate Air Filter for AirClean Workstations 
			Storage Temperature and Conditions: Room temperature</td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Thermo Fisher</td>
			<td>06666A</td>
			<td>Kimberly-Clark Professional Kimtech Science Kimwipes Delicate Task Wipers, 1-Ply 
			Storage Temperature and Conditions: Room temperature</td>
		</tr>
		<tr>
			<td>Loading Trough</td>
			<td>Thermo Fisher</td>
			<td>14387069</td>
			<td>Thermo Scientific Matrix Reagent Reservoirs 
			Storage Temperature and Conditions: Room temperature</td>
		</tr>
		<tr>
			<td>Microsoft Excel</td>
			<td>Microsoft</td>
			<td>Not Applicable</td>
			<td>Microsoft 365 package, Excel software application 
			Storage Temperature and Conditions: Room temperature</td>
		</tr>
		<tr>
			<td>Mini Centrifuge</td>
			<td>Genesee Scientific</td>
			<td>31-500B</td>
			<td>Poseidon 31-500B Mini Centrifuge, Blue Lid 
			Storage Temperature and Conditions: Room temperature</td>
		</tr>
		<tr>
			<td>PCR Grade H2O</td>
			<td>Thermo Fisher</td>
			<td>AM9937</td>
			<td>Nuclease-Free Water (not DEPC-Treated) 
			Storage Temperature and Conditions: Room temperature</td>
		</tr>
		<tr>
			<td>PCR Hood</td>
			<td>USA Scientific</td>
			<td>4263-2588</td>
			<td>Nucleic Acid Workstation with HEPA Filtration, AirClean Systems Combination PCR Workstation 
			Storage Temperature and Conditions: Room temperature</td>
		</tr>
		<tr>
			<td>PCR Strips</td>
			<td>Thermo Fisher</td>
			<td>AB0776</td>
			<td>Low Profile Tubes and Flat Caps, Strips of 8 
			Storage Temperature and Conditions: Room temperature</td>
		</tr>
		<tr>
			<td>PCR Tube Rack</td>
			<td>Thermo Fisher</td>
			<td>344820</td>
			<td>Fisherbrand 96-Well PCR Tube Rack 
			Storage Temperature and Conditions: Room temperature</td>
		</tr>
		<tr>
			<td>Pipette Tips (Multichannel)</td>
			<td>Ranin</td>
			<td>17005860</td>
			<td>Pipette Tips SR LTS 20&#181;L F 960A/5, 20&#181;L Maximum 
			Storage Temperature and Conditions: Room temperature</td>
		</tr>
		<tr>
			<td>Pipette Tips (Single Channel)</td>
			<td>USA Scientific</td>
			<td>1181-3850</td>
			<td>10&#181;L Graduated TipOne RPT Filter Tips 
			Storage Temperature and Conditions: Room temperature</td>
		</tr>
		<tr>
			<td>Pipette Tips (Single Channel)</td>
			<td>USA Scientific</td>
			<td>1180-1850</td>
			<td>20&#181;L Beveled TipOne RPT Filter Tips 
			Storage Temperature and Conditions: Room temperature</td>
		</tr>
		<tr>
			<td>Pipette Tips (Single Channel)</td>
			<td>USA Scientific</td>
			<td>1111-0880</td>
			<td>200&#181;L Natural TipOne Pipette Tips in Racks 
			Storage Temperature and Conditions: Room temperature</td>
		</tr>
		<tr>
			<td>Pipette Tips (Single Channel)</td>
			<td>USA Scientific</td>
			<td>1111-2890</td>
			<td>1000&#181;L Natural Graduated TipOne Pipette Tips in Racks 
			Storage Temperature and Conditions: Room temperature</td>
		</tr>
		<tr>
			<td>Pipettors (Multichannel)</td>
			<td>Ranin</td>
			<td>17013802</td>
			<td>Pipet-Lite Multi Pipette L8-10XLS, 0.5 to 10&#181;L 
			Storage Temperature and Conditions: Room temperature</td>
		</tr>
		<tr>
			<td>Pipettors (Multichannel)</td>
			<td>Ranin</td>
			<td>17013803</td>
			<td>Pipet-Lite Multi Pipette L8-20LS+, 2 to 20&#181;L 
			Storage Temperature and Conditions: Room temperature</td>
		</tr>
		<tr>
			<td>Pipettors (Single Channel)</td>
			<td>Thermo Fisher</td>
			<td>F144802G</td>
			<td>Gilson Pipetman Classic Pipets, 1 to 10&#181;L 
			Storage Temperature and Conditions: Room temperature</td>
		</tr>
		<tr>
			<td>Pipettors (Single Channel)</td>
			<td>Thermo Fisher</td>
			<td>F123600</td>
			<td>Gilson Pipetman Classic Pipets, 2 to 20&#181;L 
			Storage Temperature and Conditions: Room temperature</td>
		</tr>
		<tr>
			<td>Pipettors (Single Channel)</td>
			<td>Thermo Fisher</td>
			<td>F123601</td>
			<td>Gilson Pipetman Classic Pipets, 20 to 200&#181;L 
			Storage Temperature and Conditions: Room temperature</td>
		</tr>
		<tr>
			<td>Pipettors (Single Channel)</td>
			<td>Thermo Fisher</td>
			<td>F123602</td>
			<td>Gilson Pipetman Classic Pipets, 200 to 1000&#181;L 
			Storage Temperature and Conditions: Room temperature</td>
		</tr>
		<tr>
			<td>Plate Sealing Film</td>
			<td>Bio-Rad</td>
			<td>MSB1001</td>
			<td>Microseal &#8220;B&#8221; PCR Plate Sealing Film, Adhesive, Optical 
			Storage Temperature and Conditions: Room temperature</td>
		</tr>
		<tr>
			<td>Plate Spinner</td>
			<td>Thermo Fisher</td>
			<td>14-100-141</td>
			<td>Fisherbrand Mini Plate Spinner Centrifuge, 230 V 
			Storage Temperature and Conditions: Room temperature</td>
		</tr>
		<tr>
			<td>Pre-Hood Filter</td>
			<td>USA Scientific</td>
			<td>4235-3724</td>
			<td>Prefilter for AirClean Systems Workstations 
			Storage Temperature and Conditions: Room temperature</td>
		</tr>
		<tr>
			<td>R Software</td>
			<td>The R Project for Statistical Computing</td>
			<td>Not Applicable</td>
			<td>R version 4.2.2 
			Storage Temperature and Conditions: Room temperature</td>
		</tr>
		<tr>
			<td>Scale</td>
			<td>Thermo Fisher</td>
			<td>01-922-329</td>
			<td>OHAUS 30430060 PR Series Analytical Balance, 62g Capacity 
			Storage Temperature and Conditions: Room temperature</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Office Depot</td>
			<td>458612</td>
			<td>Office Depot Brand Scissors, 8&#8221;, Straight, Black, Pack of 2 
			Storage Temperature and Conditions: Room temperature</td>
		</tr>
		<tr>
			<td>Sharpies</td>
			<td>Sharpie</td>
			<td>2151734</td>
			<td>Brush Twin Permanent Markers, Black 
			Storage Temperature and Conditions: Room temperature</td>
		</tr>
		<tr>
			<td>Single Copy Gene Forward Primer</td>
			<td>Integrated DNA Technologies</td>
			<td>Custom</td>
			<td>See separate table 
			Storage Temperature and Conditions: minus 20 &#176;C when rehydrated</td>
		</tr>
		<tr>
			<td>Single Copy Gene Reverse Primer</td>
			<td>Integrated DNA Technologies</td>
			<td>Custom</td>
			<td>See separate table 
			Storage Temperature and Conditions: minus 20 &#176;C when rehydrated</td>
		</tr>
		<tr>
			<td>Small Tube Rack</td>
			<td>Thermo Fisher</td>
			<td>21-402-17</td>
			<td>Thermo Fisher 8601 Reversible Microtube Racks with Lid 
			Storage Temperature and Conditions: Room temperature</td>
		</tr>
		<tr>
			<td>Sodium Acetate</td>
			<td>Thermo Fisher</td>
			<td>J63560.EQE</td>
			<td>3M NaOAc pH 5.2 
			Storage Temperature and Conditions: Room temperature</td>
		</tr>
		<tr>
			<td>Stainless Steel Spatula</td>
			<td>Thermo Fisher</td>
			<td>3990240</td>
			<td>Bel-Art SP Scienceware Stainless-Steel Sampling Spoon and Spatula 
			Storage Temperature and Conditions: Room temperature</td>
		</tr>
		<tr>
			<td>SYBR Green</td>
			<td>Thermo Fisher</td>
			<td>S7563</td>
			<td>SYBR Green I Nucleic Acid Gel Stain &#8211; 10,000X Concentrate in DMSO 
			Storage Temperature and Conditions: minus 20 &#176;C when rehydrated</td>
		</tr>
		<tr>
			<td>Syringe Filters</td>
			<td>Fisher Scientific</td>
			<td>09-927-55A</td>
			<td>GD/X 25 mm Sterile Syringe Filter, cellulose acetate filtration medium, 0.2 &#956;m 
			Storage Temperature and Conditions: Room temperature</td>
		</tr>
		<tr>
			<td>Syringes</td>
			<td>Thermo Fisher</td>
			<td>148232A</td>
			<td>BD Luer-Lok Disposable Syringes without Needles, 10mL 
			Storage Temperature and Conditions: Room temperature</td>
		</tr>
		<tr>
			<td>TE Buffer</td>
			<td>Fisher Scientific</td>
			<td>BP2474100</td>
			<td>TE Buffer, Tris-EDTA, 1X Solution, pH 7.6, Molecular Biology, Fisher BioReagents 
			Storage Temperature and Conditions: Room temperature</td>
		</tr>
		<tr>
			<td>Telomere Forward Primer</td>
			<td>Integrated DNA Technologies</td>
			<td>Custom</td>
			<td>See separate table 
			Storage Temperature and Conditions: minus 20 &#176;C when rehydrated</td>
		</tr>
		<tr>
			<td>Telomere Reverse Primer</td>
			<td>Integrated DNA Technologies</td>
			<td>Custom</td>
			<td>See separate table 
			Storage Temperature and Conditions: minus 20 &#176;C when rehydrated</td>
		</tr>
		<tr>
			<td>UV Light</td>
			<td>USA Scientific</td>
			<td>4288-2540</td>
			<td>UV Light Bulb for Workstations 
			Storage Temperature and Conditions: Room temperature</td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td>Thermo Fisher</td>
			<td>14-955-151</td>
			<td>Fisherbrand Mini Vortex Mixer, 115 V, 50/60 Hz 
			Storage Temperature and Conditions: Room temperature</td>
		</tr>
		<tr>
			<td>Weigh Boat</td>
			<td>Thermo Fisher</td>
			<td>01-549-752</td>
			<td>Fisherbrand Sterile Hexagonal Weighing Boat, 10mL 
			Storage Temperature and Conditions: Room temperature</td>
		</tr>
	</tbody>
</table>

monochrome multiplex quantitative pcr telomere length measurement 

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&#160;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.

Author Spotlight: Exploring the Impact of Trauma on Cellular Aging

Monochrome Multiplex Quantitative PCR Telomere Length Measurement 

Telomeres are repetitive nucleoprotein regions at chromosome ends that shorten each time cells divide, resulting in decreases in average telomere length across the lifespan. Our lab investigates the long-term and intergenerational impacts of trauma and adversity on cellular aging by measuring telomere length and population-based studies. Interest in telomere biology is expanding beyond measuring average telomere length.New methods are being developed to assess chromosome specific telomere length and investigate links between health outcomes and other aspects of telomere biology, like telomere associated proteins and DNA damage. The interdisciplinary Telomere Research Network led by laboratory PI, Dr.Stacy Drury, works to enhance telomere measurement rigor and reproducibility by supporting international collaborative studies documenting the impact of pre-analytic factors on telomere assay precision, as well as establishing guidelines for the reporting of telomere length measurements generated using QPCR. The enhanced precision of MMQPCR allows for more granular analyses of the impacts of early adversity and life course environmental exposure on cellular aging, for example, by identifying periods of sensitivity where in exposures that occur during this time window produced more profound impacts on later life health outcomes.

The results presented in Table 4 and Table 5 offer an example of highly repeatable TL measurements obtained by following the protocol. For these results, DNA was extracted from 24 peripheral blood mononuclear cell (PBMC) samples using a commercial kit per manufacturers&#39; guidelines. These 24 samples were run across two 96-well plates. All DNA samples were checked for quality via spectrophotometer and fluorometer, using the average 260/280 ratio, average 260/230 ratio, and dsDNA concentration to determine assay eligibility and sample dilution factor (Table 6). Table 6 also highlights the importance of quantifying dsDNA concentration, which can differ from DNA concentration measured using a spectrophotometer. This variability is a result of different approaches to quantification. Specifically, the spectrophotometer derives DNA concentration based on absorption at 280 nm and is susceptible to fluctuations due to contaminants (e.g., protein, salt, etc.) impacting the absorbance readings. By contrast, dsDNA concentrations measured using a fluorometer are determined by the fluorescence of a dye that specifically binds to dsDNA and, as such, are presumed to be a more accurate reflection of DNA content. The DNA samples experienced up to three freeze-thaw cycles prior to MMqPCR TL analysis. The control DNA was created from pooled DNA extractions of PBMCs from one individual, which was used to create a seven-point serial dilution from 2 ng/&#956;L to 0.0313 ng/&#956;L of DNA. The independent standard curves created for PCR Step 9 (telomere amplicon) and Step 12 (single copy gene amplicon, albumin in this protocol) are presented in Figure 6A,B. The assays were run on a commercial Real-Time PCR Detection System which generated a melt curve that shows the individual amplicon products being produced at distinct temperatures as seen in Figure 8.
Table 4: Output data from MMqPCR measurement for optimal representative results. Mean T/S ratios, SDs, CVs, and Z-scored TL for 24 samples. <a href="https://www.jove.com/files/ftp_upload/66545/Table 4_RE.xlsx" target="_blank">Please click here to download this Table.</a>
Table 5: Telomere length data output. Raw data from the MMqPCR assay is analyzed in the software then added to the spreadsheet template attached as Supplementary File 1&#160;for further analysis and QC. This TL template output sheet presents a summary of the data, displaying sample IDs, average TLs, SDs, and CVs&#160;for each sample across both plates. <a href="https://www.jove.com/files/ftp_upload/66545/Table 5_RE.xlsx" target="_blank">Please click here to download this Table.</a>
Table 6: Spectrophotometer and Fluorometer DNA quality metrics for sample results. QC&#160;data from duplicate spectrophotometer analysis and singular fluorometer measurement of dsDNA and any contaminates per sample. <a href="https://www.jove.com/files/ftp_upload/66545/Table 6_RE.xlsx" target="_blank">Please click here to download this Table.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/66545/66545fig08.jpg" /> 
Figure 8: Example melt curve. Melt curve for sample data as generated in the software. The earlier peak ~80 &#176;C represents the telomere amplicon and the secondary peak at ~89 &#176;C represents the albumin amplicon. <a href="https://www.jove.com/files/ftp_upload/66545/66545fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
For this project, the average telomere efficiency was 98.6% with a range of 90.7 to 102.1 and the average albumin efficiency was 102.3% with a range of 93.6 to 108.2 across all runs. There was an average of 0.97 replicates removed from the standard curves, which meets the QC criteria of this protocol. The average interplate CV was 1.83% with a SD of 0.00616 and the average intraplate CV was 3.78% with a SD of 0.00658. The average TLs for these representative samples are presented in Table 4 and&#160;Table 5. The mean TL was 1.37 with an SD of 0.24 and a range of 0.84 to 2.32. T/S ratio, SD, CV, and Z-scored TL per sample are listed in Table 4. Since the T/S ratio output of the&#160;MMqPCR assay is a relative measurement of TL, the ratios were transformed into this Z-score to allow for cross-study comparison. ICC for this project was calculated using R script as described by TRN guidelines found in Supplementary File 2, accounting for batch and run effects20. In order to calculate the intra-project ICC, we re-ran 10% of the passing samples, making sure to populate the ICC plates with at least one sample from each plate of the cohort. The overall project ICC of 0.801 [CI: 0.703, 0.86] indicates the high reproducibility of TL results.
Not all results will be optimal. The results in Figure 9&#160;and Table 7&#160;show suboptimal results from the MMqPCR assay. Figure 9&#160;displays a standard curve with a telomere efficiency below 90%, which is below QC standards, requiring the entire plate to be repeated. Problems with primer efficiencies are usually due to an issue with reagents, so it is important to track the dates reagents are aliquoted and when they expire as a first step in determining which reagent is responsible for low efficiency. Expired reagents should be replaced before the plate is run again. Table 7&#160;displays a plate that passed initial sample-level QC criteria but has a high level of variability between the plates, leading to plate-level QC failure. Variation between plates is usually due to errors in pipetting and plate-filling techniques. In this case, the technician should evaluate any issues that occurred during plate set up and ensure pipettes are calibrated.
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/66545/66545fig09.jpg" /> 
Figure 9: Suboptimal results. This standard curve displayed in the software failed to pass QC, since the efficiency of amplification for the telomere primer was less than 90%. The image is from P1, but P2 had similarly low efficiencies. The data from this run could not be used and all samples needed to be run again after replacing the causal reagent that was expired. <a href="https://www.jove.com/files/ftp_upload/66545/66545fig09large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 7: Suboptimal Results. The table displays the telomere data template for a run where many of the samples failed to meet QC standards. Samples that needed to be re-run were determined based on CV values then the sample name was changed to a red font for easy identification. <a href="https://www.jove.com/files/ftp_upload/66545/Table 7_RE.xlsx" target="_blank">Please click here to download this Table.</a>
Supplementary File 1: Telomere data spreadsheet template. <a href="https://www.jove.com/files/ftp_upload/66545/Supplemental File 1. MMqPCR Template_RE.zip" target="_blank">Please click here to download this File.</a>
Supplementary File 2: ICC calculation. This protocol has been created by the Telomere Research Network (TRN). This file has been modified from20. <a href="https://www.jove.com/files/ftp_upload/66545/Supplemental File 2. ICC Calculation_RE.zip" target="_blank">Please click here to download this File.</a>
Supplementary File 3: TRN reporting guidelines. <a href="https://www.jove.com/files/ftp_upload/66545/Supplemental File 3. TRN Reporting Guidelines_RE.zip" target="_blank">Please click here to download this File.</a>

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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. ...

monochrome-multiplex-quantitative-pcr-telomere-length-measurement

Research

JoVE Journal

Genetics

Sample and Master Mix Preparation for Telomere Length Measurement Using Monochrome Multiplex Quantitative PCR (MMqPCR)

Begin by adding genomic DNA samples extracted from peripheral blood mononuclear cells to PCR strips A, B, and C respectively. Then vortex the thawed controlled DNA aliquot for 30 seconds. After briefly centrifuging the tube, aspirate the full volume into the first tube in the standard curve PCR tube strip.Next, add 70 microliters of PCR grade water into the second through eight tubes of the standard curve PCR strip. Then resuspend control DNA in the first tube and aspirate 70 microliters into the second tube. Wait for 30 seconds and resuspend the solution in the second tube.Collect the master mix reagents and allow them to reach room temperature in the PCR hood. Then add one microliter of the cyber green aliquot to the buffer aliquot. Vortex all aliquots for 10 seconds and centrifuge them for five seconds.Then add the specified amounts of all master mix reagents and primers into the five milliliter betaine tube. After taking out the DNA polymerase from minus 20 degrees Celsius, vortex it for 10 seconds, and then centrifuge for five seconds. Once centrifuged, slowly add 128 microliters to the master mix.Vortex the five milliliter master mix tube for 30 seconds and then set it aside.

Preparation of 96-Well Plate for Monochrome Multiplex Quantitative PCR (MMqPCR) for Telomere Length Measurement

To begin, place two 96-well plates, a loading trough, and multichannel pipette tips inside the PCR hood. After labeling each plate, turn plate 1 180 degrees so that the column numbers are upside down while leaving plate 2 facing the technician. Then pour the five milliliter tube of the prepared MM qPCR master mix into the loading trough.Use a pipet tip to ensure all the solution from the tube is transferred. Attach tips to the multichannel pipette, pressing at the base of each tip to secure a tight seal. Using reverse pipetting, dispense the master mix into either all even or all odd columns first, alternating between the two plates.After vortexing and centrifuging the PCR strips, arrange them in the tube rack in the order they will be placed on the plates. Now turn both plates 180 degrees so that the column numbers are reversed in relation to the technician. Use the multichannel pipette to resuspend solutions in the PCR strips.Using reverse pipetting, fill the first three columns of each plate with PCR strip A, alternating between even or odd columns like the master mix placement. Using a 200 microliter pipette set to 90 microliters, thoroughly resuspend the seventh standard dilution tube in PCR strip S and fill the fourth through sixth columns on the plates. After filling the plates, gently tap them on the bench top to ensure the liquid in the wells settles at the bottom.Then cover the tops of the plates with sealing films, pressing down on all edges of the film with fingertips to ensure a tight seal. To mix the contents of the plates, swirl them on the hood top for 30 seconds. Then place the sealed plates in the plate spinner for two minutes with the well openings facing the center.Now place the plates in the thermocycler ensuring the column numbers are legible. Clean the top of each plate with a clean tissue before closing the thermocycler top. Then click the Start Run button on the thermocycler software for both machines.When prompted, enter a title for the analysis files, which will be used to label and track the data from this experiment.

Monochrome Multiplex Quantitative PCR (MMqPCR) Data Analysis to Check the Reproducibility of Telomere Length Measurements

Begin by performing the MM QPCR assay for telomere length measurement. Then open the software and select the plate setup feature. After that, choose the view or edit plate option from the dropdown menu.Now highlight all the wells on the plate. Click select floraphores. Check the box labeled cyber, and ensure all other boxes are unchecked.Then click okay to confirm. While maintaining the highlight on all wells, locate and check the box next to cyber, adjacent to the word load to label all the wells with cyber. Now highlight the three non template control wells positioned either at the top or bottom of the standard control serial dilution.Then from the sample type menu on the right, select NTC. Highlight the 21 wells that make up the standard control serial dilution, and choose standard from the sample type menu. While these wells are still selected, click on technical replicate, in the replicate size menu, choose three.Then select horizontal and click apply. After that, scroll down to the dilution series section and enter two in the dilution factor field. For the P1 plate, input two times 10 to the power of negative three in the starting concentration field.Then select the box for decreasing and click apply to confirm the settings. For the P2 plate, enter 3.13 times 10 to the power of negative five in the starting concentration field. Ensure to check the box for increasing and then select apply to finalize the setup.Now highlight the columns corresponding to PCR strip A from the sample type menu, choose unknown, then proceed to select technical replicate. In the replicate size menu, pick three and opt for horizontal before clicking apply. Click okay at the bottom right of the plate editor window.Confirm the changes by clicking yes when prompted. Next, verify that the curves in the quantification tab appear appropriate for both step nine and step 12, and that the efficiency values between these two steps do not diverge by more than 10%For P1, select step nine and highlight the 21 wells corresponding to the standard curve. Copy the CQ values from the lower right corner of the software window.Then open the Telomere data sheet template and paste these values into the standard one sheet column B.Afterward, input the slope value of step nine into cell C four in the standard one sheet, check that the coefficient of variation for each standard dilution triplicate is below 0.1. On CFX, exclude any outlier data points from the analysis that make a set of triplicates fall out of range. Now, confirm that only the 72 sample wells in the P1 analysis file are highlighted in blue in the quantification tab.Then in step nine, copy the CQ and SQ values found in the lower right corner of the software window and paste them into the samples P1 sheet beginning at cell D3.Using CFX maestro, ensure all CQ values for analytical samples are within the range of the lowest and highest CQ values of the standard curve. In the Excel sheet, verify that the starting concentration in the NTC is less than 5%of the average amount of DNA in the sample wells. For sample level quality control, examine the standard deviations and coefficient of variations in columns J and K on the samples P1 and P2 sheets.Verify that the individual sample interplate CVs in the P1 versus P2 sheet, specifically in column G, do not exceed 0.05. If a sample's interplate CV is higher than acceptable, remove up to one triplicate from the calculation for each sample. For plate level quality control, check that the average interplate variation across all samples on each plate is less than 0.05.If a sample's interplate CV is higher than acceptable, remove up to one triplicate from the calculation for each sample. For additional plate level quality control, check that the overall interplate variation in P1 versus P2 sheet is less than 0.06.

722 Views.  Tulane University.  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.

Accurate Telomere Length Assessment in Human Population Studies

Summary