Novel Bioprinting Application for the Production of Reference Material Containing a Defined Copy Number of Target DNA

Seo, Manabu; Takabatake, Reona; Izumi, Satoshi; Unno, Hirotaka; Kawashima, Yudai; Ki, Unoh; Hatada, Shigeo; Katoh, Ikuo; Nakazawa, Satoshi; Matsumoto, Takahiko; Yonekawa, Yuuki; Hashimoto, Michie; Lin, Waka; Maeda, Reo; Riztyan,; Onishi, Mari; Futo, Satoshi; Kishine, Masahiro; Kitta, Kazumi

doi:10.1021/acs.analchem.9b02207

Cited by 7 publications

(10 citation statements)

References 20 publications

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“…We considered the specified number of copies to be dispensed as a measured value and calculated the uncertainty of measurements from the production variability and the number of samples used. In addition to the variability of the copy number of intracellular target DNA and the variability because of contamination, which have been evaluated in the previous study 13 , cell aggregation was identified to be another source of the production variability. To increase the production efficiency, two avalanche photodiodes (APD410A/M, Thorlabs; hereinafter referred to as APD) placed oppositely were used to detect any cells in flying droplets (Figure S1 in Supplementary Information File 1).…”

Section: Resultsmentioning

confidence: 96%

“…The intracellular DNA copy number distribution was measured in the same manner as in the previous study 13 , although a flow cytometer (MA900, Sony) was used for the measurement. In flow cytometry, a cell containing two nuclei cannot be distinguished from an aggregate of two cells containing one nucleus per cell.…”

Section: Resultsmentioning

confidence: 99%

“…The preparations of cell suspensions for preparation of the new reference material and treatments after dispensing, such as cell wall lysis, were performed as described in the previous study 13 , except that Synthetic construct DNA 6203-a-G (600-G, GenBank registration number AB610938.1), instead of hmg-Le1 , was incorporated into yeast.…”

Section: Methodsmentioning

confidence: 99%

“…This can be interpreted to indicate that the performance of the reaction system should be assessed using calibrators (reference nucleic acid materials) covering a full range of concentrations to be quantified to obtain more reliable analysis results from quantitative real-time PCR 10 , 11 analysis with extremely small amounts of nucleic acids 12 . To meet such demand, we developed a new cell-based DNA reference material (hereinafter referred to as the new reference material) containing the target DNA sequence as low as one copy 13 . Note that “copy” is a unit generally used to express one DNA molecule containing a certain sequence.…”

Section: Introductionmentioning

confidence: 99%

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Evaluation method for asymmetric uncertainty of quantitative polymerase chain reaction measurements of deoxyribonucleic acids with low copy number

Suzuki

Nakazawa

et al. 2021

Sci Rep

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Recently, in food safety and various other fields, qualitative and quantitative gene analysis using real-time polymerase chain reaction (PCR) method has become increasingly popular. The limit of detection (LOD) and quantifiable range for these measurements depends on the range and precision of DNA calibrators’ concentrations. Low-copy-number nucleic acid reference materials with low uncertainty produced by an inkjet system have been developed to allow for precise measurements in a low-copy-number region. However, when using a calibrator with a low copy number near one, the copy number distribution is asymmetric. Consequently, the confidence intervals of estimated copy numbers can include negative values when conventional methods of uncertainty estimation are used. A negative confidence interval is irrelevant in the context of copy number, which is always positive value or zero. Here, we propose a method to evaluate the uncertainty of real-time PCR measurements with representative values and an asymmetric 95% confidence interval. Moreover, we use the proposed method for the actual calculation of uncertainty of real-time PCR measurement results for low-copy-number DNA samples and demonstrate that the proposed method can evaluate the precision of real-time PCR measurements more appropriately in a low-copy-number region.

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Section: Resultsmentioning

confidence: 96%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Evaluation method for asymmetric uncertainty of quantitative polymerase chain reaction measurements of deoxyribonucleic acids with low copy number

Suzuki

Nakazawa

et al. 2021

Sci Rep

Self Cite

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show abstract

“…In order to develop an RNA quantification method that can measure up to one copy of RNA, a standard product in which RNA is dispensed as one copy per well is required. In recent years, although not regarding RNA, standard products have been developed in which DNA is dispensed as one copy/well [104]. In this method, cells are discharged into the well while controlling the number of cells with an inkjet head, instead of dispensing the DNA itself into the well.…”

Section: Methods To Dispense the Solution More Accuratelymentioning

confidence: 99%

Challenges for Accurate Quantification of RNA

Hasegawa

Hapsari

Iwahashi

2021

RAS

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Ribonucleic acid (RNA) quantification is an essential technique in biology. There has been remarkable progress in RNA quantification techniques over the recent years; however, the specificity of these techniques to quantify a very small amount of RNA is doubtful because of factors which can inhibit precise quantification. To develop a technique that leads to the most sensitive RNA quantification, these problems must be overcome. In this article, we first review the factors that inhibit precise quantification of RNA: the quality of RNA, secondary structure of RNA, efficiency of the enzyme reaction, annealing conditions, limitations of the experimental protocol and equipment, and detection sensitivity of the equipment. Next, we discuss the possible methods which contribute to these factors: RNA quality control focused on target RNA degradation, isothermal amplification, techniques for avoiding amplification errors, RNase H-dependent PCR, targeting using a fluorescent-labeled probe, targeting using a padlock probe, bridged/locked nucleic acid (BNA/LNA) and peptide nucleic acid (PNA), and the clustered regularly interspaced short palindromic repeat (CRISPR) system. One of the goals for the development of an ultrasensitive RNA quantification technique is the absolute quantification of RNA. Here, we discuss the techniques used for this type of RNA quantification.

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Development of gene-in-plasmid DNA reference materials certified by single-molecule counting

Lee,

Yoo,

Hong

et al. 2024

Anal Bioanal Chem

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The mole, the SI unit for measuring the amount of a substance, was redefined as a fixed number of entities. This definition enables straightforward quantification of substances by counting individual entities. Counting proves particularly effective for quantifying large and discrete biological entities such as DNA, proteins, viruses, and cells, which are challenging to quantify via traditional physical or chemical methods. In this study, we detail our approach to develop gene reference materials certified through single-molecule counting, which enables mole-traceable measurements. We quantified three plasmid DNA constructs, each carrying a specific gene of interest, via single-molecule counting. The resulting values were cross-validated via digital PCR and LC‒MS. Sequence impurities in the certified reference materials were quantified via single-molecule real-time sequencing, whereas fragment impurities were quantified via two-color digital PCR analysis. We precisely accounted for various sources of uncertainty, including measurement precision, weighing, homogeneity, and impurities, when estimating the total uncertainty of the reference materials. In conclusion, a practical format for gene-based DNA reference materials, a measurement method to achieve metrological traceability, and methods for quantifying fragments and sequence impurities were developed and implemented in this study. We anticipate that our gene-based DNA reference materials will serve as valuable higher-order standards for the calibration of other methods or reference materials for DNA quantification in a variety of bioanalytical applications.

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Novel Bioprinting Application for the Production of Reference Material Containing a Defined Copy Number of Target DNA

Cited by 7 publications

References 20 publications

Evaluation method for asymmetric uncertainty of quantitative polymerase chain reaction measurements of deoxyribonucleic acids with low copy number

Evaluation method for asymmetric uncertainty of quantitative polymerase chain reaction measurements of deoxyribonucleic acids with low copy number

Challenges for Accurate Quantification of RNA

Development of gene-in-plasmid DNA reference materials certified by single-molecule counting

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