sample and master mix preparation for telomere length measurement using monochrome multiplex quantitative pcr (mmqpcr)

Accurate Telomere Length Assessment in Human Population Studies

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>

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.

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.

sample-master-mix-preparation-for-telomere-length-measurement-using

Research

JoVE Journal

Genetics

128 Views. 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 microlite...

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

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.

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. ...

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.