Comparison of Suitability of the Most Common Ancient DNA Quantification Methods

Brzobohatá, Kristýna; Drozdová, Eva; Smutny, Jiri; Zeman, Tomáš; Beňuš, Radoslav

doi:10.1089/gtmb.2016.0197

Cited by 12 publications

(7 citation statements)

References 41 publications

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“…This approach has been extensively used for microRNA analysis of plasma samples . The estimation of the isolated samples has been performed by NanoDrop Nucleic Acid Quantification procedure described by Brzobohatá et al (2016) . For each RNA extraction, 800 μL of plasma were added with 2400 μL TRIzol LS.…”

Section: Methodsmentioning

confidence: 99%

Altered erythroid‐related miRNA levels as a possible novel biomarker for detection of autologous blood transfusion misuse in sport

et al. 2019

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BACKGROUND: Autologous blood transfusion (ABT) is a performance-enhancing method prohibited in sport; its detection is a key issue in the field of anti-doping. Among novel markers enabling ABT detection, microRNAs (miRNAs) might be considered a promising analytical tool. STUDY DESIGN AND METHODS:We studied the changes of erythroid-related microRNAs following ABT, to identify novel biomarkers. Fifteen healthy trained males were studied from a population of 24 subjects, enrolled and randomized into a Transfusion (T) and a Control (C) group. Seriated blood samples were obtained in the T group before and after the two ABT procedures (withdrawal, with blood refrigerated or cryopreserved, and reinfusion), and in the C group at the same time points. Traditional hematological parameters were assessed. Samples were tested by microarray analysis of a pre-identified set of erythroid-related miRNAs. RESULTS:Hematological parameters showed moderate changes only in the T group, particularly following blood withdrawal. Among erythroid-related miRNAs tested, following ABT a pool of 7 miRNAs associated with fetal hemoglobin and regulating transcriptional repressors of gamma-globin gene was found stable in C and differently expressed in three out of six T subjects in the completed phase of ABT, independently from blood conservation. Particularly, two or more erythropoiesis-related miRNAs within the shortlist constituted of miR- 126-3p, miR-144-3p, miR-191-3p, miR-197-3p, miR-486-3p, miR-486-5p, and miR-92a-3p were significantly upregulated in T subjects after reinfusion, with a person-to-person variability but with congruent changes. CONCLUSIONS:This study describes a signature of potential interest for ABT detection in sports, based on the analysis of miRNAs associated with erythroid features.A utologous blood transfusion (ABT) is a performance-enhancing method prohibited in sport, 1 with its detection a key issue in the anti-doping field. Given the lack of a method for its direct detection, 2-5 an approach to the indirect detection of ABT has been considered. The athlete biological passport (ABP) hematological module 6-8 has been adopted by the World Anti-Doping Agency (WADA) to identify and, when necessary, sanction athletes showing abnormal changes in hematological parameters. Novel markers enabling ABT indirect detection have also been proposed, [2][3][4][9][10][11][12][13] including those derived from genomics applied to microRNAs (miRNAs), and are considered a promising analytical tool. [14][15][16][17] In this respect, miRNAs are short non-coding RNA molecules, which act as gene regulators by repressing translation or by inducing the cleavage of target RNA transcripts. [18][19][20][21] Recent evidence suggests that miRNAs (including miRNAs present as extracellular molecules in plasma) can be considered a From the

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Section: Methodsmentioning

confidence: 99%

Altered erythroid‐related miRNA levels as a possible novel biomarker for detection of autologous blood transfusion misuse in sport

et al. 2019

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“…Many studies have previously compared different NGS library quantification methods and shown contradictory results (Brzobohatá et al, 2017;Hussing et al, 2015;Katsuoka et al, 2014;Laurie et al, 2013;Nakayama et al, 2016;Robin et al, 2016;White et al, 2009). Hussing with colleagues quantified dsDNA oligos and revealed that BioAnalyzer, TapeStation, and Qubit instruments give concentrations closest to the expected (Hussing et al, 2015).…”

Section: Resultsmentioning

confidence: 99%

Ultralow Coverage Sequencing Is the Most Accurate Library Quantification Method Prior to Exome Sequencing

Krasnenko¹,

Stetsenko²,

Rakitko³

et al. 2019

PTQ

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Accurate DNA library quantification is very important in post-pooling captured exome sequencing. Underrepresented libraries will need additional sequencing, which takes extra time and money, whereas overexpressed DNA libraries can lead to the generation of more data than required, which leads to the waste of sequence capacity and a reduced number of samples per batch. There is a number of methods available to quantify DNA libraries prior to sequencings, such as UV absorption, use of intercalating dyes, capillary electrophoresis, 5’-hydrolysis probes (TaqMan©) coupled with quantitative PCR (qPCR) or droplet digital PCR. But there is no gold standard for the quantification of DNA libraries. This study compares common library quantification methods, including LabChip (PerkinElmer Inc., MA, USA), Qubit 3.0 (Thermo Fisher Scientific, MA, USA), several qPCR approaches, and ultralow coverage sequencing on Illumina MiSeq platform (with and without insert size correction). DNA libraries were prepared using the NEBNext Ultra DNA Library Prep Kit for Illumina (New England Biolabs, MA, USA). To compare the above-mentioned approaches, cost, time, and quantification accuracy were assessed in our study. Quantification methods involving the use of Qubit and MiSeq were found to be better than qPCR and LabChip approaches at predicting the final library concentration. It was also revealed that MiSeq with insert size correction was the most accurate method for library quantification prior to exome sequencing. This method allows for correction shifts in the ratio due to enrichment. Ultralow coverage sequencing on the Illumina MiSeq platform is the most accurate method of library quantification prior to pooling and post-pooling exome enrichment.

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“…Real-time polymerase chain reactions did not allow for concentration estimation by under-measurement. The most reliable method is fragment analysis, which helps to determine the concentration of DNA and the length of a given fragment [78].…”

Section: Extraction Of Ancient Dnamentioning

confidence: 99%

Methodological Changes in the Field of Paleogenetics

Danielewski

Żuraszek

Zielińska

et al. 2023

Genes

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Paleogenetics has significantly changed since its inception almost forty years ago. Initially, molecular techniques available to the researchers offered minimal possibilities for ancient DNA analysis. The subsequent expansion of the scientific tool cabinet allowed for more remarkable achievements, combined has with the newfound popularity of this budding field of science. Finally, a breakthrough was made with the development of next-generation sequencing (NGS) technologies and the update of DNA isolation protocols, through which even very fragmented aDNA samples could be used to sequence whole genomes. In this paper, we review the achievements made thus far and compare the methodologies utilized in this field of science, discussing their benefits and challenges.

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Comparison of Suitability of the Most Common Ancient DNA Quantification Methods

Cited by 12 publications

References 41 publications

Altered erythroid‐related miRNA levels as a possible novel biomarker for detection of autologous blood transfusion misuse in sport

Altered erythroid‐related miRNA levels as a possible novel biomarker for detection of autologous blood transfusion misuse in sport

Ultralow Coverage Sequencing Is the Most Accurate Library Quantification Method Prior to Exome Sequencing

Methodological Changes in the Field of Paleogenetics

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