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.remotexs.ntu.edu.sg/files/ftp_upload/66545/66545fig01largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>0.5mL Tubes</td><td>USA Scientific</td><td>1605-0099</td><td>Seal-Rite 0.5mL Microcentrifuge Tubes, Sterile&lt;br/&gt; 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&lt;br/&gt; 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 H&lt;sub&gt;2&lt;/sub&gt;O&lt;br/&gt; Storage Temperature and Conditions: minus 20 &amp;deg;C&amp;nbsp;</td></tr><tr><td>15mL Tubes</td><td>Thermo Fisher</td><td>14-959-53A</td><td>Corning 352196 Falcon 15mL Conical Centrifuge Tubes&lt;br/&gt; Storage Temperature and Conditions: Room temperature</td></tr><tr><td>1M MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Thermo Fisher</td><td>50152107</td><td>Biotang Inc 1M MgCl&lt;sub&gt;2&lt;/sub&gt; 1M Magnesium Chloride Solution, Prepared in 18.2 Megohms Water and Filtered through 0.22 Micron Filter&lt;br/&gt; Storage Temperature and Conditions: 4 &amp;deg;C&amp;nbsp;</td></tr><tr><td>1x Gold Buffer</td><td>In House</td><td>Not Applicable</td><td>10X Gold Buffer diluted with PCR Grade H&lt;sub&gt;2&lt;/sub&gt;O&lt;br/&gt; Storage Temperature and Conditions: minus 20 &amp;deg;C&amp;nbsp;</td></tr><tr><td>25mM dNTPs</td><td>New England BioLabs</td><td>N0446S</td><td>Deoxynucleotide Solution Set&lt;br/&gt; Storage Temperature and Conditions: minus 20 &amp;deg;C&amp;nbsp;</td></tr><tr><td>5mL Tubes</td><td>Thermo Fisher</td><td>3391276</td><td>Argos Technologies Microcentrifuge Tubes &amp;ndash; 5mL&lt;br/&gt; 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&lt;br/&gt; 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&quot; x 75&#039;, Silver&lt;br/&gt; Storage Temperature and Conditions: Room temperature</td></tr><tr><td>AmpliTaq Gold Kit &amp;ndash; Polymerase and Buffer</td><td>Thermo Fisher</td><td>4311806</td><td>AmpliTaq Gold DNA Polymerase with Gold Buffer and MgCl&lt;sub&gt;2&lt;/sub&gt; (MgCl&lt;sub&gt;2&lt;/sub&gt; in this kit is not used), 10X Gold Buffer, 2.5U AmpliTaq Gold Polymerase&lt;br/&gt; Storage Temperature and Conditions: minus 20 &amp;deg;C&amp;nbsp;</td></tr><tr><td>Betaine</td><td>Thermo Fisher</td><td>AAJ77507AB</td><td>Betaine, 5M Solution, Molecular Biology Grade, Ultrapure, 10mL&lt;br/&gt; Storage Temperature and Conditions: minus 20 &amp;deg;C&amp;nbsp;</td></tr><tr><td>Big Tube Rack</td><td>Thermo Fisher</td><td>344817</td><td>Fisherbrand 4-Way Tube Rack&lt;br/&gt; 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&lt;br/&gt; 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&lt;br/&gt; Storage Temperature and Conditions: Room temperature</td></tr><tr><td>DTT</td><td>Fisher Scientific</td><td>AAJ1539706</td><td>Dithiothreitol, &gt;99.5+ Molecular Biology Grade, 5 g&lt;br/&gt; Storage Temperature and Conditions: minus 20 &amp;deg;C&amp;nbsp;</td></tr><tr><td>ELIMINase</td><td>Fisher Scientific</td><td>04-355-32</td><td>ELIMINase Laboratory Decontaminant&lt;br/&gt; 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&lt;br/&gt; 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&lt;br/&gt; 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&lt;br/&gt; 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&lt;br/&gt; 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&lt;br/&gt; Storage Temperature and Conditions: Room temperature</td></tr><tr><td>PCR Grade H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Thermo Fisher</td><td>AM9937</td><td>Nuclease-Free Water (not DEPC-Treated)&lt;br/&gt; 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&lt;br/&gt; 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&lt;br/&gt; 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&lt;br/&gt; 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&amp;micro;L F 960A/5, 20&amp;micro;L Maximum&lt;br/&gt; 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&amp;micro;L Graduated TipOne RPT Filter Tips&lt;br/&gt; 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&amp;micro;L Beveled TipOne RPT Filter Tips&lt;br/&gt; 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&amp;micro;L Natural TipOne Pipette Tips in Racks&lt;br/&gt; 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&amp;micro;L Natural Graduated TipOne Pipette Tips in Racks&lt;br/&gt; 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&amp;micro;L&lt;br/&gt; 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&amp;micro;L&lt;br/&gt; 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&amp;micro;L&lt;br/&gt; 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&amp;micro;L&lt;br/&gt; 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&amp;micro;L&lt;br/&gt; 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&amp;micro;L&lt;br/&gt; Storage Temperature and Conditions: Room temperature</td></tr><tr><td>Plate Sealing Film</td><td>Bio-Rad</td><td>MSB1001</td><td>Microseal &amp;ldquo;B&amp;rdquo; PCR Plate Sealing Film, Adhesive, Optical&lt;br/&gt; 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&lt;br/&gt; 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&lt;br/&gt; 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&lt;br/&gt; 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&lt;br/&gt; 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&amp;rdquo;, Straight, Black, Pack of 2&lt;br/&gt; Storage Temperature and Conditions: Room temperature</td></tr><tr><td>Sharpies</td><td>Sharpie</td><td>2151734</td><td>Brush Twin Permanent Markers, Black&lt;br/&gt; 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&lt;br/&gt; Storage Temperature and Conditions: minus 20 &amp;deg;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&lt;br/&gt; Storage Temperature and Conditions: minus 20 &amp;deg;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&lt;br/&gt; 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&lt;br/&gt; 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&lt;br/&gt; 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 &amp;ndash; 10,000X Concentrate in DMSO&lt;br/&gt; Storage Temperature and Conditions: minus 20 &amp;deg;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 &amp;mu;m&lt;br/&gt; 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&lt;br/&gt; 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&lt;br/&gt; 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&lt;br/&gt; Storage Temperature and Conditions: minus 20 &amp;deg;C when rehydrated</td></tr><tr><td>Telomere Reverse Primer</td><td>Integrated DNA Technologies</td><td>Custom</td><td>See separate table&lt;br/&gt; Storage Temperature and Conditions: minus 20 &amp;deg;C when rehydrated</td></tr><tr><td>UV Light</td><td>USA Scientific</td><td>4288-2540</td><td>UV Light Bulb for Workstations&lt;br/&gt; 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&lt;br/&gt; 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&lt;br/&gt; 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.

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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/files/ftp_upload/66545/Supplemental File 3. TRN Reporting Guidelines_RE.zip" target="_blank">Please click here to download this File.</a>

Read this Scientific Article Protocol about Author Spotlight: Exploring the Impact of Trauma on Cellular Aging at JoVE.com

Author Spotlight: Exploring the Impact of Trauma on Cellular Aging

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.

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. ...

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

Nanyang Technological University

Research

JoVE Journal

Genetics

1.5K Views.  Tulane University. Telomeren zijn repetitieve nucleoproteïnegebieden aan chromosoomuiteinden die elke keer dat cellen zich delen verkorten, wat resulteert in een afname van de gemiddelde telomeerlengte gedurende de levensduur. Ons laboratorium onderzoekt de langetermijn- en intergenerationele effecten van trauma en tegenspoed op cellulaire veroudering door telomeerlengte en populatiegebaseerde studies te meten. De belangstelling voor telomeerbiologie gaat verder dan het meten ...

Video: Auteur in de schijnwerpers: onderzoek naar de impact van trauma op cellulaire veroudering

Improved Polymerase Chain Reaction-restriction Fragment Length Polymorphism Genotyping of Toxic Pufferfish by Liquid Chromatography/Mass Spectrometry

An improved version of a polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method for genotyping toxic pufferfish species by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) is described. DNA extraction is carried out using a silica membrane-based DNA extraction kit. After the PCR amplification using a detergent-free PCR buffer, restriction enzymes are added to the solution without purifying the reaction solution. A reverse-phase silica monolith column and a Fourier transform high resolution mass spectrometer having a modified Kingdon trap analyzer are employed for separation and detection, respectively. The mobile phase, consisting of 400 mM 1,1,1,3,3,3-hexafluoro-2-propanol, 15 mM triethylamine (pH 7.9) and methanol, is delivered at a flow rate of 0.4 ml/min. The cycle time for LC/ESI-MS analysis is 8 min including equilibration of the column. Deconvolution software having an isotope distribution model of the oligonucleotide is used to calculate the corresponding monoisotopic mass from the mass spectrum. For analysis of oligonucleotides (range 26-79 nucleotides), mass accuracy was 0.62 &#177; 0.74 ppm (n = 280) and excellent accuracy and precision were sustained for 180 hr without use of a lock mass standard.

An improved polymerase chain reaction-restriction fragment length polymorphism method for genotyping pufferfish species by liquid chromatography/mass spectrometry is described. A reverse-phase silica monolith column is employed for separating digested amplicons. This method can elucidate the monoisotopic masses of oligonucleotides, which is useful for identifying base composition.

An improved version of a polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method for genotyping toxic pufferfish species by ...

Merging Absolute and Relative Quantitative PCR Data to Quantify STAT3 Splice Variant Transcripts

Human signal transducer and activator of transcription 3 (STAT3) is one of many genes containing a tandem splicing site. Alternative donor splice sites 3 nucleotides apart result in either the inclusion (S) or exclusion (&#916;S) of a single residue, Serine-701. Further downstream, splicing at a pair of alternative acceptor splice sites result in transcripts encoding either the 55 terminal residues of the transactivation domain (&#945;) or a truncated transactivation domain with 7 unique residues (&#946;). As outlined in this manuscript, measuring the proportions of STAT3's four spliced transcripts (S&#945;, S&#946;, &#916;S&#945; and &#916;S&#946;) was possible using absolute qPCR (quantitative polymerase chain reaction). The protocol therefore distinguishes and measures highly similar splice variants. Absolute qPCR makes use of calibrator plasmids and thus specificity of detection is not compromised for the sake of efficiency. The protocol necessitates primer validation and optimization of cycling parameters. A combination of absolute qPCR and efficiency-dependent relative qPCR of total STAT3 transcripts allowed a description of the fluctuations of STAT3 splice variants' levels in eosinophils treated with cytokines. The protocol also provided evidence of a co-splicing interdependence between the two STAT3 splicing events. The strategy based on a combination of the two qPCR techniques should be readily adaptable to investigation of co-splicing at other tandem splicing sites.

Tandem splicing events occur at sites less than 12 nucleotides apart. Quantifying ratios of such splice variants is feasible using an absolute quantitative PCR approach. This manuscript describes how splice variants of the gene STAT3, in which two splicing events results in Serine-701 inclusion/exclusion and &#945;/&#946; C-termini, can be quantified.

Human signal transducer and activator of transcription 3 (STAT3) is one of many genes containing a tandem splicing site. Alternative donor splice sites 3 ...

Single-cell Gene Expression Using Multiplex RT-qPCR to Characterize Heterogeneity of Rare Lymphoid Populations

Gene expression heterogeneity is an interesting feature to investigate in lymphoid populations. Gene expression in these cells varies during cell activation, stress, or stimulation. Single-cell multiplex gene expression enables the simultaneous assessment of tens of genes1,2,3. At the single-cell level, multiplex gene expression determines population heterogeneity4,5. It allows for the distinction of population heterogeneity by determining both the probable mix of diverse precursor stages among mature cells and also the diversity of cell responses to stimuli.
Innate lymphoid cells (ILC) have been recently described as a population of innate effectors of the immune response6,7. In this protocol, cell heterogeneity of the ILC hepatic compartment is investigated during homeostasis.
Currently, the most widely used technique to assess gene expression is RT-qPCR. This method measures gene expression only one gene at a time. Additionally, this method cannot estimate heterogeneity of gene expression, since multiple cells are needed for one test. This leads to the measurement of the average gene expression of the population. When assessing large numbers of genes, RT-qPCR becomes a time-, reagent-, and sample-consuming method. Hence, the trade-offs limit the number of genes or cell populations that can be evaluated, increasing the risk of missing the global picture.
This manuscript describes how single-cell multiplex RT-qPCR can be used to overcome these limitations. This technique has benefited from recent microfluidics technological advances1,2. Reactions occurring in multiplex RT-qPCR chips do not exceed the nanoliter-level. Hence, single-cell gene expression, as well as simultaneous multiple gene expression, can be performed in a reagent-, sample-, and cost-effective manner. It is possible to test cell gene signature heterogeneity at the clonal level between cell subsets within a population at different developmental stages or under different conditions4,5. Working on rare populations with large numbers of conditions at the single-cell level is no longer a restriction.

This protocol describes how to assess the expression of a large array of genes at the clonal level. Single-cell RT-qPCR produces highly reliable results with a strong sensitivity for hundreds of samples and genes.

Gene expression heterogeneity is an interesting feature to investigate in lymphoid populations. Gene expression in these cells varies during cell ...

Modified Terminal Restriction Fragment Analysis for Quantifying Telomere Length Using In-gel Hybridization

There are several different techniques for measuring telomere length, each with their own advantages and disadvantages. The traditional approach, Telomere Restriction Fragment (TRF) analysis, utilizes a DNA hybridization technique whereby genomic DNA samples are digested with restriction enzymes, leaving behind telomere DNA repeats and some sub-telomeric DNA. These are separated by agarose gel electrophoresis, transferred to a filter membrane and hybridized to oligonucleotide probes tagged with either chemiluminescence or radioactivity to visualize telomere restriction fragments. This approach, while requiring a larger quantity of DNA than other techniques such as PCR, can measure the telomere length distribution of a population of cells and allows measurement expressed in absolute kilobases. This manuscript demonstrates a modified DNA hybridization procedure for determining telomere length. Genomic DNA is first digested with restriction enzymes (that do not cut telomeres) and separated by agarose gel electrophoresis. The gel is then dried and the DNA is denatured and hybridized in situ to a radiolabeled oligonucleotide probe. This in situ hybridization avoids loss of telomere DNA and improves signal intensity. Following hybridization, the gels are imaged utilizing phosphor screens and the telomere length is quantified using a graphing program. This procedure was developed by the laboratories of Drs. Woodring Wright and Jerry Shay at the University of Texas Southwestern1,2. Here, we present a detailed description of this procedure, with some modifications.

In this article, a detailed protocol for quantifying telomere length using a modified terminal restriction fragment analysis is discussed that provides fast and efficient direct measurement of telomere length. This technique can be applied to a variety of cell sources of DNA for quantifying telomere length.

There are several different techniques for measuring telomere length, each with their own advantages and disadvantages. The traditional approach, Telomere ...

Cancer Research

Detection of microRNA Expression in Peritoneal Membrane of Rats Using Quantitative Real-time PCR

MicroRNAs (miRNAs) are small noncoding RNAs that regulate messenger RNA expression post-transcriptionally. The miRNA expression profile has been investigated in various organs and tissues in rat. However, standard methods for the purification of miRNAs and detection of their expression in rat peritoneal membrane have not been well established. We have developed an effective and reliable method to purify and quantify miRNAs using quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) in rat peritoneal membrane. This protocol consists of four steps: 1) purification of peritoneal membrane sample; 2) purification of total RNA including miRNA from peritoneal membrane sample; 3) reverse transcription of miRNA to produce cDNA; and 4) qRT-PCR to detect miRNA expression. Using this protocol, we successfully determined that the expression of six miRNAs (miRNA-142-3p, miRNA-21-5p, miRNA-221-3p, miRNA-223-3p, miRNA-327, and miRNA-34a-5p) increased significantly in the peritoneal membrane of a rat peritoneal fibrosis model compared with those in control groups. This protocol can be used to study the profile of miRNA expression in the peritoneal membrane of rats in many pathological conditions.

Here we present a protocol for the detection of microRNA expression in rat peritoneal membrane using quantitative real-time reverse-transcription polymerase chain reaction. This method is suitable for studying the microRNA expression profile in rat peritoneal membrane in several pathological conditions.

MicroRNAs (miRNAs) are small noncoding RNAs that regulate messenger RNA expression post-transcriptionally. The miRNA expression profile has been ...

Bidirectional Retroviral Integration Site PCR Methodology and Quantitative Data Analysis Workflow

Integration Site (IS) assays are a critical component of the study of retroviral integration sites and their biological significance. In recent retroviral gene therapy studies, IS assays, in combination with next-generation sequencing, have been used as a cell-tracking tool to characterize clonal stem cell populations sharing the same IS. For the accurate comparison of repopulating stem cell clones within and across different samples, the detection sensitivity, data reproducibility, and high-throughput capacity of the assay are among the most important assay qualities. This work provides a detailed protocol and data analysis workflow for bidirectional IS analysis. The bidirectional assay can simultaneously sequence both upstream and downstream vector-host junctions. Compared to conventional unidirectional IS sequencing approaches, the bidirectional approach significantly improves IS detection rates and the characterization of integration events at both ends of the target DNA. The data analysis pipeline described here accurately identifies and enumerates identical IS sequences through multiple steps of comparison that map IS sequences onto the reference genome and determine sequencing errors. Using an optimized assay procedure, we have recently published the detailed repopulation patterns of thousands of Hematopoietic Stem Cell (HSC) clones following transplant in rhesus macaques, demonstrating for the first time the precise time point of HSC repopulation and the functional heterogeneity of HSCs in the primate system. The following protocol describes the step-by-step experimental procedure and data analysis workflow that accurately identifies and quantifies identical IS sequences.

This manuscript describes the experimental procedure and software analysis for a bidirectional integration site assay that can simultaneously analyze upstream and downstream vector-host junction DNA. Bidirectional PCR products can be used for any downstream sequencing platform. The resulting data are useful for a high-throughput, quantitative comparison of integrated DNA targets.

Integration Site (IS) assays are a critical component of the study of retroviral integration sites and their biological significance. In recent retroviral ...

High-resolution Melting PCR for Complement Receptor 1 Length Polymorphism Genotyping: An Innovative Tool for Alzheimer's Disease Gene Susceptibility Assessment

Complement receptor 1 (CR1), a transmembrane glycoprotein that plays a key role in the innate immune system, is expressed on many cell types, but especially on red blood cells (RBCs). As a receptor for the complement components C3b and C4b, CR1 regulates the activation of the complement cascade and promotes the phagocytosis of immune complexes and cellular debris, as well as the amyloid-beta (A&#946;) peptide in Alzheimer's disease (AD). Several studies have confirmed AD-associated single nucleotide polymorphisms (SNPs), as well as a copy-number variation (CNV) in the CR1 gene. Here, we describe an innovative method for determining the length polymorphism of the CR1 receptor. The receptor includes three domains, called long homologous repeats (LHR)-LHR-A, LHR-C, and LHR-D-and an n domain, LHR-B, where n is an integer between 0 and 3. Using a single pair of specific primers, the genetic material is used to amplify a first fragment of the LHR-B domain (the variant amplicon B) and a second fragment of the LHR-C domain (the invariant amplicon). The variant amplicon B and the invariant amplicon display differences at five nucleotides outside of the hybridization areas of said primers. The numbers of variant amplicons B and of invariant amplicons is deduced using a quantitative tool (high-resolution melting (HRM) curves), and the ratio of the variant amplicon B to the invariant amplicon differs according to the CR1 length polymorphism. This method provides several advantages over the canonical phenotype method, as it does not require fresh material and is cheaper, faster, and therefore applicable to larger populations. Thus, the use of this method should be helpful to better understand the role of CR1 isoforms in the pathogenesis of diseases such as AD.

Here, we describe an innovative method to determine complement receptor 1 (CR1) length polymorphisms for use in several applications, particularly the assessment of susceptibility to diseases such as Alzheimer&#39;s disease (AD). This method could be useful to better understand the role of CR1 isoforms in the pathogenesis of AD.

Complement receptor 1 (CR1), a transmembrane glycoprotein that plays a key role in the innate immune system, is expressed on many cell types, but ...

Dissection of Enhancer Function Using Multiplex CRISPR-based Enhancer Interference in Cell Lines

Multiple enhancers often regulate a given gene, yet for most genes, it remains unclear which enhancers are necessary for gene expression, and how these enhancers combine to produce a transcriptional response. As millions of enhancers have been identified, high-throughput tools are needed to determine enhancer function on a genome-wide scale. Current methods for studying enhancer function include making genetic deletions using nuclease-proficient Cas9, but it is difficult to study the combinatorial effects of multiple enhancers using this technique, as multiple successive clonal cell lines must be generated. Here, we present Enhancer-i, a CRISPR interference-based method that allows for functional interrogation of multiple enhancers simultaneously at their endogenous loci. Enhancer-i makes use of two repressive domains fused to nuclease-deficient Cas9, SID and KRAB, to achieve enhancer deactivation via histone deacetylation at targeted loci. This protocol utilizes transient transfection of guide RNAs to enable transient inactivation of targeted regions and is particularly effective at blocking inducible transcriptional responses to stimuli in tissue culture settings. Enhancer-i is highly specific both in its genomic targeting and its effects on global gene expression. Results obtained from this protocol help to understand whether an enhancer is contributing to gene expression, the magnitude of the contribution, and how the contribution is affected by other nearby enhancers.

This protocol describes the steps needed to design and perform multiplexed targeting of enhancers with the deactivating fusion protein SID4X-dCas9-KRAB, also known as enhancer interference (Enhancer-i). This protocol enables the identification of enhancers that regulate gene expression and facilitates the dissection of relationships between enhancers regulating a common target gene.

Multiple enhancers often regulate a given gene, yet for most genes, it remains unclear which enhancers are necessary for gene expression, and how these ...

Quantifying Telomere Length Using Non-Radioactive Chemiluminescence Detection

Optimization of Performance Parameters of the TAGGG Telomere Length Assay

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.

Author Spotlight: Optimization of Performance Parameters of the TAGGG Telomere Length Assay  

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.

Telomeres are repetitive sequences which are present at chromosomal ends; their shortening is a characteristic feature of human somatic cells. Shortening ...

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

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

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

Telomeres  are repeating DNA sequences at the tip ends of the chromosomes that are diverse  in length and in humans can reach a length of 15,000 base ...

Monochrome Multiplex Quantitative PCR Telomere Length Measurement

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