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 in 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 wherein exposures that occur during this time window produce more profound impacts on later life health outcomes. Begin by adding genomic DNA samples extracted from peripheral blood mononuclear cells to PCR strips, A, B, and C respectively. Then vortex the thawed control 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 SYBR 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. To begin place two 96 well plates, a loading trough, and multichannel pipette tips inside the PCR hood.

After labeling each plate, turn plate one 180 degrees so that the column numbers are upside down while leaving plate two facing the technician. Then pour the five milliliter tube of the prepared MMqPCR master mix into the loading trough. Use a pipette 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.

Begin by performing the MMqPCR 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 fluorophores. Check the box labeled SYBR, and ensure all other boxes are unchecked.

Then click OK to confirm. While maintaining the highlight on all wells, locate and check the box next to SYBR, adjacent to the word Load, to label all the wells with SYBR. 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 OK 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 C4 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.