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

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.

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

Nucleic acid amplification methods, such as polymerase chain reaction (PCR), are extensively used in many applications to detect target DNA because of their high sensitivity, good reproducibility, and wide dynamic range of quantification. However, analytical quality control when detecting low copy number target DNA is often missing because of a lack of appropriate reference materials. Recent advances in analytical sciences require a method to accurately quantify DNA at the single molecule level. Herein, we have developed a novel method to produce reference material containing a defined copy number of target DNA (referred to as "cell number-based DNA reference material"). In this method, a suspension of cells carrying a single target DNA sequence was ejected by an inkjet head, and the number of cells in each droplet was counted using highly sensitive cameras. The resulting solutions contained a defined copy number of target DNA and could be used as reference materials. The use of the newly developed reference material was compared with that of diluted solutions of target DNA to evaluate the performance of qualitative real-time PCR in terms of the limit of detection (LOD). Our results demonstrated that cell number-based DNA reference material provides more accurate information regarding performance quality. The reference material produced by this method is a promising tool to evaluate assay performance.

High-precision three-dimensional inkjet technology for live cell bioprinting.

In recent years, bioprinting has emerged as a promising technology for the construction of three-dimensional (3D) tissues to be used in regenerative medicine or in vitro screening applications. In the present study, we present the development of an inkjet-based bioprinting system to arrange multiple cells and materials precisely into structurally organized constructs. A novel inkjet printhead has been specially designed for live cell ejection. Droplet formation is powered by piezoelectric membrane vibrations coupled with mixing movements to prevent cell sedimentation at the nozzle. Stable drop-on-demand dispensing and cell viability were validated over an adequately long time to allow the fabrication of 3D tissues. Reliable control of cell number and spatial positioning was demonstrated using two separate suspensions with different cell types printed sequentially. Finally, a process for constructing stratified Mille-Feuille-like 3D structures is proposed by alternately superimposing cell suspensions and hydrogel layers with a controlled vertical resolution. The results show that inkjet technology is effective for both two-dimensional patterning and 3D multilayering and has the potential to facilitate the achievement of live cell bioprinting with an unprecedented level of precision.