Name: Video: Preparation of 96-Well Plate for Monochrome Multiplex Quantitative PCR (MMqPCR) for Telomere Length Measurement
Uploaded: 2024-03-22T14:50:00
Duration: 2 min 41 s
Description: 82 Views. Preparation of 96-Well Plate for Monochrome Multiplex Quantitative PCR (MMqPCR) for Telomere Length Measurement

preparation of 96-well plate for monochrome multiplex quantitative pcr (mmqpcr) for telomere length measurement

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>

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Monochrome Multiplex Quantitative PCR Telomere Length Measurement

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

preparation-96-well-plate-for-monochrome-multiplex-quantitative-pcr

Research

JoVE Journal

Genetics

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

Video: Preparation of 96-Well Plate for Monochrome Multiplex Quantitative PCR (MMqPCR) for 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.

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. ...