Impact of Amplification Efficiency Approaches on Telomere Length Measurement via Quantitative-Polymerase Chain Reaction

Background: Precise determination of amplification efficiency is critical for reliable conversion of within-sample changes in fluorescence occurring on a logarithmic scale to between-sample differences in DNA content occurring on a linear scale. This endeavor is especially challenging for the telomere length (TL) quantitative-PCR (qPCR) assay, where amplification efficiency can vary between reactions targeting telomeric repeats (T) and those targeting a single-copy gene (S) to calculate TL as the T/S ratio.Methods: We compared seven different approaches toward estimating amplification efficiency, including the standard-curve method utilized by the qPCR instrument software, and alternative approaches which estimate efficiency on a reaction-by-reaction basis using the stand-alone program LinRegPCR. After calculating T/S ratios using efficiency estimates from each approach (N = 363), we tested their relative performance on metrics of assay precision and correlates of external validity including chronological age (age range = 1–72 years), across tissues within-person (leukocyte-buccal), and between parents and offspring.Results: Estimated amplification efficiency for telomere reactions was significantly lower than estimates for single-copy gene reactions. Efficiency estimates for both reaction sets were significantly higher when estimated with the standard-curve method utilized by the qPCR instrument relative to estimates reconstructed during the log-linear phase with LinRegPCR. While estimates of single-copy gene efficiency reconstructed using LinRegPCR measured within 90% of perfect exponential doubling (E = 1.92), estimates generated using the standard-curve method were inflated beyond 100% (E = 2.10–2.12), indicating poor fidelity. Despite differences in raw value, TL measurements calculated with LinRegPCR efficiency estimates exhibited similar relationships with external validity correlates to measurements generated using the qPCR instrument software.Conclusion: Since methods to estimate amplification efficiency can vary across qPCR instruments, we suggest that future analyses empirically consider external methods of efficiency calculations such as LinRegPCR, and that already generated data be re-analyzed to glean possible improvements.

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A Synthetic Single-Stranded Dual-Template Oligonucleotide as a Reference Standard for Monochrome Multiplex qPCR Measurements of Average Telomere Length

10.1101/2020.06.24.169797 ◽

2020 ◽

Author(s):

Richard M. Cawthon

Keyword(s):

Telomere Length ◽

Quantitative Pcr ◽

Single Copy ◽

Repeat Sequence ◽

Experimental Sample ◽

Single Copy Gene ◽

Reference Standard ◽

Standard Curve ◽

Telomere Sequence ◽

Copy Gene

ABSTRACTQuantitative PCR is frequently used to measure average telomere length (TL) relative to the TL of a reference DNA sample of the investigator’s choosing. This makes comparisons of TLs across studies and laboratories difficult. Here we demonstrate that a single synthetic single-stranded dual-template oligonucleotide (DTO) containing both a telomere repeat sequence (T) and a segment of the human beta-globin (HBB) single copy gene (S) can be used as a universal reference standard for monochrome multiplex quantitative PCR (MMqPCR) measurements of average TL using SYBR Green I as the only fluorescent reporter dye. A set of twelve concentrations of the DTO is prepared by serial 3-fold dilutions, to a lowest concentration of ~20 copies per μl. The 5 highest concentrations are used for the T standard curve, and the 5 lowest concentrations are used for the S standard curve. For each reaction 5 μl containing approximately 3 ng of genomic DNA (or one of the DTO dilutions) is mixed with 5 μl of a 2x MasterMix containing the primers for T and S amplification, and MMqPCR is performed. The design of the primers and thermal cycling profile allows all T amplification signals to be collected before exponential amplification of the S signal begins. Exponential amplification from S is then carried out in a temperature range that keeps the telomere product fully melted and therefore unable to influence the S amplification signal. The T value for each DNA sample is the Standard Curve DTO dilution that contains the same number of copies of the telomere sequence as the experimental sample, and the S value is the DTO dilution that contains the same number of copies of the single copy gene sequence as the experimental sample. Dividing the first dilution by the second dilution yields an absolute T/S ratio, since it is expressed relative to the fixed 1:1 T/S ratio that is built into the DTO by design. Absolute T/S ratios for average TL in 48 human DNA samples determined by this method correlated strongly with mean Terminal Restriction Fragment (mTRF) lengths for the same DNA samples determined by the Southern Blot method (R-squared = 0.801). This DTO and the accompanying protocol may facilitate the standardization of average telomere length measurements and analyses across laboratories.

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Exceptionally Long-Lived Individuals (ELLI) Demonstrate Slower Aging Rate Calculated by DNA Methylation Clocks as Possible Modulators for Healthy Longevity

International Journal of Molecular Sciences ◽

10.3390/ijms21020615 ◽

2020 ◽

Vol 21 (2) ◽

pp. 615

Author(s):

Danielle Gutman ◽

Elina Rivkin ◽

Almog Fadida ◽

Lital Sharvit ◽

Vered Hermush ◽

...

Keyword(s):

Dna Methylation ◽

Telomere Length ◽

Single Copy ◽

Phenotypic Characterization ◽

Single Copy Gene ◽

Online Tool ◽

Healthy Longevity ◽

Relative Telomere Length ◽

Dna Methylation Profile ◽

Copy Gene

Exceptionally long-lived individuals (ELLI) who are the focus of many healthy longevity studies around the globe are now being studied in Israel. The Israeli Multi-Ethnic Centenarian Study (IMECS) cohort is utilized here for assessment of various DNA methylation clocks. Thorough phenotypic characterization and whole blood samples were obtained from ELLI, offspring of ELLI, and controls aged 53–87 with no familial exceptional longevity. DNA methylation was assessed using Illumina MethylationEPIC Beadchip and applied to DNAm age online tool for age and telomere length predictions. Relative telomere length was assessed using qPCR T/S (Telomere/Single copy gene) ratios. ELLI demonstrated juvenile performance in DNAm age clocks and overall methylation measurement, with preserved cognition and relative telomere length. Our findings suggest a favorable DNA methylation profile in ELLI enabling a slower rate of aging in those individuals in comparison to controls. It is possible that DNA methylation is a key modulator of the rate of aging and thus the ELLI DNAm profile promotes healthy longevity.

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Increased Telomere Length Is a Negative a Predicting Factor for IST in Children with Severe Aplastic Anemia?

Blood ◽

10.1182/blood.v118.21.4381.4381 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4381-4381

Author(s):

Katarzyna Pawelec ◽

Marek Janiak ◽

Michał Matysiak ◽

Pawel Krzysztof Wlodarski

Keyword(s):

Aplastic Anemia ◽

Telomere Length ◽

Peripheral Blood ◽

Genomic Dna ◽

Antithymocyte Globulin ◽

Single Copy ◽

Single Copy Gene ◽

Blood Samples ◽

Copy Gene ◽

Predicting Factor

Abstract Abstract 4381 Shortening of telomeres is observed in 1/3 of peripheral blond samples, collected from patients with severe aplastic anemia (SAA). Probably, this phenomenon is associated with poor response to immunosuppressive therapy –IST (antithymocyte globulin -ATG and cyklosporin-CSA). The aim of this study was to assess the length of telomeres in peripheral blood of children with SAA treated with antithymocyte globulin (ATG) and cyklosporin (CSA) Materials and Methods: Peripheral blood samples were collected from 13 children with confirmed SAA. Patients (9 girls and 4 boys) aged 7–19 yrs have all received rabbit ATG in a dose of 3.75 mg/kg/day for 5 days and CSA in a dose of 5mg/kg/day for 12–14 months). Remission was assessed on 180th and 360th day of treatment. Control peripheral blood samples were obtained from 12 healthy children (3 girls and 9 boys, aged 3–17 yrs), 12 healthy adultand from 4 SAA children parents. Informed consent was obtained from all adult donors and from parents of participating children. DNA extraction and qPCR Genomic DNA was extracted from whole blood using AxyPrep Blood Genomic DNA Miniprep Kit (Axygene). Telomere length was determined using the quantitive PCR (qPCR) method described by Elisa Pavesi et al (1). Briefly, two qPCR reactions were run for each DNA sample: amplification of telomere product and amplification of a single copy gene (SEP15). Each qPCR reaction contained: Power SYBR1 Green PCR Master Mix (Applied Biosystems), template DNA and primers (270nM Tel1 and 900nM Tel2 or 500nM of each SEP15 primers). Telomere/single copy gene (T/S) ratios for samples were calculated using Human reference DNA (Applied Biosystems) standard curve. Reactions were preformed in Real Time PCR CFX96 system (Biorad). Statistica software version 9.1 (StatSoft) was used for statistical analysis Results: Among 13 SAA patients telomere shortening was found in 9 children while 4 patients had significantly elongated telomeres. All of the latter ones responded poorly to IST treatment: 3 of them did not respond (NR) to IST (neither on day 180 nor on 360) and one patient had a partial remission (PR). 2 patients in the NR group were randomized to unrelated bone marrow transplantation and one had another course of IST (no unrelated donor), after which he reached a PR. All patients with elongated telomeres had normal serum adriostendion level. Telomere elongation was also found in parents compared to the control group of adults (n=12, age range 31–57). Among 9 patients with shortened telomeres, 4 achieved complete remission (CR), 1 - PR and 4 did not respond (NR) to treatment on check times points. Conclusions: Our observations indicate that SAA patients with shortened telomeres respond to immunosuppressive therapy better then those with elongated telomeres. Telomere length may be considered therefore as a predicting factor in this patients. Studies on a larger group of patients should be performed to confirm our observations. Short telomere definition –samples, which T/S value is less than the first Quartile of controls. Long telomere - samples, which T/S value is in III quartile of controls. Very long telomere - samples, which T/S value is higher than the IV Quartile of controls. Disclosures: No relevant conflicts of interest to declare.

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Association of Telomere Length of Peripheral Blood Leukocyte with Hematological Response of Immunosuppressive Therapy in Children with Severe Aplastic Anemia

Blood ◽

10.1182/blood.v120.21.4403.4403 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4403-4403 ◽

Cited By ~ 1

Author(s):

Katarzyna Pawelec ◽

Marek Janiak ◽

Michal Matysiak ◽

Malgorzata Salamonowicz ◽

Pawel Krzysztof Wlodarski

Keyword(s):

Aplastic Anemia ◽

Immunosuppressive Therapy ◽

Telomere Length ◽

Peripheral Blood ◽

Genomic Dna ◽

Single Copy ◽

Severe Aplastic Anemia ◽

Late Response ◽

Single Copy Gene ◽

Copy Gene

Abstract Abstract 4403 Introduction: Severe aplastic anemia (SAA) is characterized by life-threatening cytopenias. SAA can be cured by hematopoietic stem cell transplantation, but in children when a sibling donor is unavailable, immunosuppressive therapy with antithymocyte globulin (ATG) plus cyclosporine is effective. Variations in telomere length have been reported in severe aplastic anemia but their clinical significance is not clear, The aim of our study is relationship between telomere length and clinical response to immunosuppressive therapy children with severe aplastic anemia. Material and methods: We measured telomere length in children with SAA at our institution who had received ATG plus cyclosporine between 2009 to 2011and analyzed its relationship with hematologic recovery. Patients 18 (9 girls and 9 boys) aged 7–19 yrs with SAA without sibling donor, have all received rabbit ATG in a dose of 3.75 mg/kg/day for 5 days and CSA in a dose of 5mg/kg/day for 12 months). Remission was assessed on 84th, 112th, 180th and 360th day of treatment. Patients were divided into 3 groups: I group: non responders NR, II group: early remission -ER (complete-CR and partial remission PR on 84th and 112th day), III late remission LR (CR and PR on 180th and 360th day). We analyzed telomere length in 20 parents (11 mothers and 9 fathers aged 39–55yrs) our 11 patients. Control peripheral blood samples were obtained from 12 healthy children (3 girls and 9 boys, aged 3–17 yrs), and 10 healthy adult (aged 28–55yrs). Informed consent was obtained from all adult donors and from parents of participating children. Genomic DNA was extracted using AxyPrep Blood Genomic DNA Miniprep Kit (Axygene). Telomere length was measured using the quantitive PCR (qPCR) method described by Elisa Pavesi et al (1). Pavesi E. et al. Pediatr Blood Cancer. 2009;53(3):411-6. Two 96-well plates were prepared for measurements, first plate was used for amplification of telomere product and second one was used for single copy gene (SEP15) product amplification. Each reaction contained: Power SYBR1 Green PCR Master Mix (Applied Biosystems), DNA and primers (270nM Tel1 and 900nM Tel2 or 500nM each SEP15 primers). Reactions were preformed in Real Time PCR: CFX96 system (Biorad) with thermal cycling conditions: 95C–10 minutes; 95C–15 seconds, 54C 2 minutes (35 cycles). Telomere/single copy gene (T/S) ratios for samples were calculated from Human reference DNA (Applied Biosystems) standard curve and T/S ratios values were expressed in relative to telomere length in controls. Statistica software version 9.0 (StatSoft, Tulsa, OK) was used for statistical analysis. Mann–Whitney U test was employed to compare telomere length among samples groups. p value <0.05 was considered statistically significant. Results: The telomere length in children with SAA were shorter than health children but this not significantly to (p=0, 16) Twelve patients (66, 6%) responded to immunosuppressive therapy. There was correlation between telomere length and the late response. Children with late response were shorter telomere than children with control group (Fig 1). We not noted in our patient relapse and clonally transformation, but time of observation was only 3 year Parents children with SAA telomere length were shorter than health control adult people, but without significantly (p=0, 15) (Fig 2). We found correlation between shorter telomere length parents and late response to immunosupression therapy in children with SAA (p= 0,012) (Fig 3). Conclusions: Our observations indicate that SAA patients with shortened telomeres respond to immunosuppressive therapy better then those with elongated telomeres. Telomere length parents of SAA children with response of IST were shorter than parent's in children with SAA without response. Telomere length may be considered therefore as a predicting factor in these patients. Studies on a larger group of patients should be performed to confirm our observations. Disclosures: No relevant conflicts of interest to declare.

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Shorter peripheral blood telomeres are a potential biomarker for patients with advanced colorectal adenomas

The International Journal of Biological Markers ◽

10.5301/jbm.2012.9347 ◽

2012 ◽

Vol 27 (4) ◽

pp. 375-380 ◽

Cited By ~ 14

Author(s):

Douglas L. Riegert-Johnson ◽

Lisa A. Boardman ◽

Julia E. Crook ◽

Colleen S. Thomas ◽

Ruth A. Johnson ◽

...

Keyword(s):

Telomere Length ◽

Peripheral Blood ◽

Colorectal Neoplasia ◽

Population Group ◽

Single Copy ◽

Colorectal Adenomas ◽

Control Group ◽

Single Copy Gene ◽

Potential Biomarker ◽

Copy Gene

Background Colorectal cancer (CRC) can be prevented by the early detection and removal of advanced adenomas (AAs) by colonoscopy. Our aim was to evaluate peripheral blood leukocyte (PBL) telomere length as a potential biomarker for the presence of AAs. Methods PBL telomere length was measured in patients with AAs (n=35), in a control group of similarly aged patients who had a normal colonoscopy (n=145) and in a separate population group with no history of cancer, again similarly aged (n=495). Telomere measurements were performed using a quantitative PCR assay and reported as ratios of telomere and single copy gene measurements. Results Telomere lengths tended to be lower in patients with AAs than in patients in the normal colonoscopy group (p<0.001) as well as those in the population group (p=0.011). A telomere/single copy gene ratio of 0.5 was found to have an estimated 94% sensitivity and a 56% specificity for AAs; a combination of sensitivity and specificity for which a value of >0.5 would reduce the odds of a patient having AAs by a factor of 0.11 (the negative likelihood ratio). Thirty three percent of individuals in the population group tested above this cutoff and could be considered at low risk for AAs. Conclusions PBL telomeres are shortened in patients with colorectal neoplasia, suggesting that PBL telomere length could be a promising non-invasive blood biomarker to pre-screen for risk of AAs prior to colonoscopy.

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Identification of a single-copy gene encoding a Type I chlorophyll a/b-binding polypeptide of photosystem I in Arabidopsis thaliana

Physiologia Plantarum ◽

10.1034/j.1399-3054.1992.840410.x ◽

1992 ◽

Vol 84 (4) ◽

pp. 561-567 ◽

Cited By ~ 1

Author(s):

Poul E. Jensen ◽

Michael Kristensen ◽

Tine Hoff ◽

Jan Lehmbeck ◽

Bjarne M. Stummann ◽

...

Keyword(s):

Arabidopsis Thaliana ◽

Chlorophyll A ◽

Photosystem I ◽

Single Copy ◽

Type I ◽

Single Copy Gene ◽

Gene Encoding ◽

Copy Gene

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A Comparative Analysis of Methods for Titering Reovirus

Pharmaceutical Nanotechnology ◽

10.2174/2211738508666200826103322 ◽

2020 ◽

Vol 8 (5) ◽

pp. 409-417

Author(s):

Yi-Chen Yang ◽

Xian-Yao Wang ◽

Yuan-Yuan An ◽

Chun-Xiang Liao ◽

Nian-Xue Wang ◽

...

Keyword(s):

Traditional Method ◽

Virus Titer ◽

Cell Analysis ◽

Half Cell ◽

L929 Cells ◽

Cell Index ◽

Standard Curve ◽

Analysis Technique ◽

Curve Method ◽

Standard Curve Method

Background: A key challenge in the process of virus amplification is the need for a simple and convenient method for measuring virus titers. Objective: Real-time unlabeled cell analysis (RTCA) was used to establish a standard curve of correlation between half-cell index time (CIT50) and virus titer. At the same time, the virus titer from tunable resistance pulse detection (TRPS) technology was compared with the traditional median tissue culture infectious dose (TCID50) method to evaluate the feasibility and application value of the RTCA technique and TRPS technology. Methods: : Cell index (CI) values for L929 cells under different culture conditions were detected, and the appropriate initial cell inoculation density was screened. The half-cell index (CI50) values of reovirus infected L929 cells with TCID50 titers were analyzed by RTCA, the CI50-TCID50 standard curve was created, and a regression equation was developed. RTCA, TCID50, and TRPS methods were used to detect the reovirus titer obtained by the amplification, and the sensitivity and feasibility of the CIT50-TCID50 standard curve method were analyzed. The virus titer was detected by TRPS technology and the TCID50 method. Results: L929 cells were best propagated at an initial density of 6 × 103 cells/well. After infecting L929 cells with different titers of reference reovirus, the linear correlation of CIT50 and TCID50 was y = -2.1806x + 71.023 (R2 = 0.9742). The titer resulting from the RTCA assay was 7×109.6821 pfu/mL, from the TRPS assay was 4.52×1010 pfu/mL, and from the TCID50 assay was 7×109.467 pfu/mL. Conclusion: The CIT50-TCID50 standard curve method established by the RTCA technique can be used to quantitatively detect reovirus titer with L929 cells. Compared with the TCID50 method, it takes a relatively short time and has high sensitivity and accuracy. The TRPS technology requires even less time to quantify the virus, but its precision is lower than that of the TCID50 method and RTCA technology. This study provides new technical methods for assessing the virulence of infectious live reovirus particles. Lay Summary: After amplification of the virus, we need to detect the virus titers (the virulence of the virus). The traditional method is to use the virus to infect cells, and then the virus titers can be calculated by 50% of the cells infected. However, this traditional method is time consuming. The ways of RTCA (a real-time cell analysis technique) and TRPS (a nano-bioparticle analysis technique) help us to detect viral titers. The consistency of these three methods determines their feasibility and accuracy. If they are feasible, then these two simple technologies will provide new ideas for detecting viral titers.

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A Mouse Single-Copy Gene,Gtf2i,the Homolog of HumanGTF2I,That Is Duplicated in the Williams–Beuren Syndrome Deletion Region

Genomics ◽

10.1006/geno.1997.5182 ◽

1998 ◽

Vol 48 (2) ◽

pp. 163-170 ◽

Cited By ~ 28

Author(s):

Yu-Ker Wang ◽

Luis A. Pérez-Jurado ◽

Uta Francke

Keyword(s):

Single Copy ◽

Single Copy Gene ◽

Deletion Region ◽

Copy Gene

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Real-Time PCR for Molecular Detection of Zoonotic and Non-Zoonotic Giardia spp. in Wild Rodents

Microorganisms ◽

10.3390/microorganisms9081610 ◽

2021 ◽

Vol 9 (8) ◽

pp. 1610

Author(s):

Christian Klotz ◽

Elke Radam ◽

Sebastian Rausch ◽

Petra Gosten-Heinrich ◽

Toni Aebischer

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Gastrointestinal Disease ◽

Single Copy ◽

Marker Genes ◽

Small Rodent ◽

Analytical Sensitivity ◽

Single Copy Gene ◽

Wild Rodents ◽

Copy Gene

Giardiasis in humans is a gastrointestinal disease transmitted by the potentially zoonotic Giardia duodenalis genotypes (assemblages) A and B. Small wild rodents such as mice and voles are discussed as potential reservoirs for G. duodenalis but are predominantly populated by the two rodent species Giardia microti and Giardia muris. Currently, the detection of zoonotic and non-zoonotic Giardia species and genotypes in these animals relies on cumbersome PCR and sequencing approaches of genetic marker genes. This hampers the risk assessment of potential zoonotic Giardia transmissions by these animals. Here, we provide a workflow based on newly developed real-time PCR schemes targeting the small ribosomal RNA multi-copy gene locus to distinguish G. muris, G. microti and G. duodenalis infections. For the identification of potentially zoonotic G. duodenalis assemblage types A and B, an established protocol targeting the single-copy gene 4E1-HP was used. The assays were specific for the distinct Giardia species or genotypes and revealed an analytical sensitivity of approximately one or below genome equivalent for the multi-copy gene and of about 10 genome equivalents for the single-copy gene. Retesting a biobank of small rodent samples confirmed the specificity. It further identified the underlying Giardia species in four out of 11 samples that could not be typed before by PCR and sequencing. The newly developed workflow has the potential to facilitate the detection of potentially zoonotic and non-zoonotic Giardia species in wild rodents.

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