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Optimization of a Relative Telomere Length Assay by Monochromatic Multiplex Real- Time Quantitative PCR on the LightCycler 480  Anthony Y.Y. Hsieh, Sara.

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Presentation on theme: "Optimization of a Relative Telomere Length Assay by Monochromatic Multiplex Real- Time Quantitative PCR on the LightCycler 480  Anthony Y.Y. Hsieh, Sara."— Presentation transcript:

1 Optimization of a Relative Telomere Length Assay by Monochromatic Multiplex Real- Time Quantitative PCR on the LightCycler 480  Anthony Y.Y. Hsieh, Sara Saberi, Abhinav Ajaykumar, Kyle Hukezalie, Izabella Gadawski, Beheroze Sattha, Hélène C.F. Côté  The Journal of Molecular Diagnostics  Volume 18, Issue 3, Pages (May 2016) DOI: /j.jmoldx Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

2 Figure 1 PCR cycling profile. Schematic of the monochromatic multiplex real-time quantitative PCR cycling profile, describing each stage. Program and segment markers as referenced on LightCycler 480 raw output are shown. The circles represent fluorescence signal acquisitions. The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx ) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

3 Figure 2 MgCl2 titration melting curves. Melting curves were derived using change in relative fluorescence units (ΔRFU) per 0.2°C on various amounts of pooled human whole-blood DNA. Melting curves for the following conditions are shown: 0 (A), 1 (B), 2 (C), and 3 (D) mmol/L added MgCl2. The arrow indicates the dissociation peak of a nonspecific peak present in all conditions. The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx ) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

4 Figure 3 EDTA titration melting curves. Melting curves were derived using change in relative fluorescence units (ΔRFU) per 0.2°C on various amounts of pooled human whole-blood DNA. Melting curves for the following conditions are shown: 0 (A), 0.4 (B), 0.8 (C), 1.2 (D), and 1.6 (E) mmol/L added EDTA. The arrow indicates the dissociation peak of a nonspecific peak present in A through C. The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx ) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

5 Figure 4 PCR efficiency and peak fluorescence for MgCl2 and EDTA titrations. PCR efficiencies for both acquisitions were calculated using a single 8-point standard curve (R2 > 0.99). PCR efficiencies for MgCl2 (A) and EDTA (B) titrations. Peak fluorescence was defined by the relative fluorescence unit (RFU) at the 40th cycle for both acquisitions and both internal controls (ICs). Peak fluorescence values for MgCl2 (C) and EDTA (D) titrations. Each data point represents a single DNA sample. LT, long telomere; ST, short telomere. The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx ) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

6 Figure 5 Visualization of amplicons on 3% agarose gel. Lanes 1 and 9 were loaded with a 100-bp ladder. Lanes 2 through 6 are PCR products after the standard 40-cycle profile. Lane 2 is the long telomere internal control (LT-IC) with all primers, lane 3 is the short telomere internal control (ST-IC) with all primers, and lane 4 is negative control (using DNA elution buffer) with all primers. Lane 5 is the LT-IC with only ALB primers, and lane 6 is the LT-IC with only telomere primers. Lanes 7 and 8 are PCR products after only 30 cycles, with the rest of the program kept the same. Lane 7 is the LT-IC with all primers, and lane 8 is negative control (DNA elution buffer) with all primers. All PCR products were diluted 1:10 before loading onto the agarose gel. The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx ) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

7 Figure 6 Telomere and ALB product melting curves after 25 and 40 cycles. Melting curves were derived using change in relative fluorescence units (ΔRFU) per 0.2°C on long telomere internal control (LT-IC) and short telomere internal control (ST-IC) after 25 (A) and 40 (B) cycles. The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx ) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

8 Figure 7 Minimum quantifiable telomere/single-copy nuclear gene (T/S) ratio and amplification with individual primer pairs. A: T/S ratios of long telomere internal control (LT-IC) were calculated using standard curves prepared from placenta DNA, a tissue with LTs, spiked with varying quantities of a plasmid containing the ALB amplicon (without GC clamps). Theoretical standard curve T/S ratios are shown on the x axis and IC T/S ratios on the y axis. IC T/S ratios were from two runs, each containing six technical replicates. The vertical dotted line marks the minimum quantifiable T/S ratio, which is 6. B: Threshold cycle (CT) values of both acquisitions are shown when the reaction contained the following primer combinations: albu+albd+telg+telc, albu+albd only and telg+telc only. Data points are derived from six to eight technical replicates. Error bars indicate SDs. The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx ) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

9 Figure 8 Limit of quantification. The telomere/single-copy nuclear gene (T/S) ratios of various amounts of pooled human whole-blood DNA were measured. Each data point represents the CV calculated from the T/S ratio of 10 technical replicates. The arrow indicates the concentration of the limit of quantification: approximately 160 pg of DNA per reaction. The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx ) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

10 Figure 9 Pearson's correlation between flow fluorescence in situ hybridization (FISH) and monochromatic multiplex real-time quantitative PCR (MMqPCR). Telomere length (TL) in cord blood samples (n = 33) were measured by flow FISH, MMqPCR, and monoplex qPCR. A: Correlations between mean MMqPCR shorter leukocyte TL (LTL) and median flow FISH lymphocyte and granulocyte TL. B: Correlations between monoplex qPCR LTL and flow FISH lymphocyte and granulocyte TL. Pearson's R2 is shown. P < for all. The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx ) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

11 Figure 10 Pearson's Correlation between terminal restriction fragment (TRF) and real-time quantitative PCR (qPCR). Telomere length (TL) in placenta (n = 21) and HT-29 cell culture (n = 36) were measured by TRF Southern blot and qPCR. TRF data were compared with monochromatic multiplex qPCR (MMqPCR) (A) and monoplex (B) qPCR. Pearson's R2 is shown. P <  for all. The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx ) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

12 Figure 11 Pearson's correlation between monoplex real-time quantitative PCR (qPCR) and monochromatic multiplex qPCR (MMqPCR). TL in dried blood spot samples (n = 58) and cord blood samples (n = 26) were measured by monoplex qPCR and MMqPCR. Two techniques were correlated on telomere/single-copy nuclear gene (T/S) (A), T (B), and S (C), using both sample sets combined (n = 84). Pearson's R2 is shown. P <  for all. The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx ) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

13 Supplemental Figure S1 Fluorescence curves of long and short telomere specimens. Fluorescence curves of both acquisitions of a long telomere placenta (A) and a short telomere cord blood (B) specimen. The circles represent the threshold cycle of the 74°C acquisition.T/S, telomere/single-copy nuclear gene. The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx ) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

14 Supplemental Figure S2 Impact of small fluctuations in standard curves. A and B: Both acquisitions of two very linear standard curves are given as examples. Log [S] on the x axis is derived from the logarithm of albumin copy numbers of each standard. Pearson's R2 and PCR efficiencies (Eff) are given. C: Telomere/single-copy nuclear gene (T/S) ratios from the DNA-independent standard curve in A. D: Small fluctuations in the standard curve of drive DNA dependence in the T/S ratio (B). Differences in PCR efficiency between the two acquisitions are given. CT, threshold cycle. The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx ) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

15 Supplemental Figure S3 Plate position effects. A long telomere internal control was measured in every well on 96-well plates, except well D7, which was used as a negative template control. A: The absolute difference in CV between the telomere/single-copy nuclear gene (T/S) ratio of each well and the plate mean is shown. This difference represents a roughly normal distribution, with approximately half of the wells deviating above the plate mean. The mean of the data from each column and row are given. Variability across columns (B) and rows (C) are shown. Error bars represent SDs calculated from the number of replicates in each column (n = 12) or each row (n = 8). Columns or rows significantly different from plate mean (using one-sample t-test) are marked, with P values and effect sizes shown. Mean effect sizes are given as the percentage difference of each row or column compared with the mean of the plate. These data are derived from the mean of three runs. The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx ) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

16 Supplemental Figure S4 Pearson's correlation between monoplex and monochromatic multiplex real-time quantitative PCR (MMqPCR). Telomere length in cord blood (n = 26) and dried blood spot (n = 58) samples were measured by monoplex and MMqPCR. Correlations between the two techniques were determined using cord blood on telomere/single-copy nuclear gene (T/S) (A), T (B), and S (C) and using dried blood spots on T/S (D), T (E), and S (F). Pearson's R2 is shown. P <  for all. The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx ) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

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