Development and interlaboratory validation of a linearized plasmid DNA certified reference material by single molecule direct counting and digital PCR.

Digital PCR (dPCR) technology is widely utilized for various applications, including the quantification of gene mutations and copy number variations. Certified reference materials (CRMs) play a critical role in improving the comparability of dPCR results, establishing SI-traceable copy number concentration values for dPCR calibration remains a key challenge due to the limited availability of CRMs value-assigned by higher-order, independent methods. To tackle this issue, a linearized plasmid DNA reference material (RM) was developed and rigorously characterized through an interlaboratory comparison involving three national measurement institutes (NMIs) from China (NIM), South Korea (KRISS), and Japan (NMIJ).

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

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.

Use of a sample injection loop for an accurate measurement of particle number concentration by flow cytometry.

Flow cytometry plays a pivotal role in biotechnology by providing quantitative measurements for a wide range of applications. Nonetheless, achieving precise particle quantification, particularly without relying on counting beads, remains a challenge. In this study, we introduce a novel exhaustive counting method featuring a sample loop-based injection system that delivers a defined sample volume to a detection system to enhance quantification in flow cytometry.

Precise RNA Quantification by Counting Individual RNA Molecules Using High-Sensitivity Capillary Flow Cytometry.

Precise determination of ribonucleic acid (RNA) concentration without the need for calibration was pursued by sequence-specific counting of individual RNA molecules. This approach eliminates the reverse transcription (RT) step required for polymerase chain reaction (PCR)-based quantification, which may hamper accurate measurements owing to uncertain yields of RT reactions. Target RNAs were tagged with a number of fluorescent oligonucleotide probes with complementary sequences.

High-sensitivity microvolume UV absorption spectrometry for routine analysis of small-volume biological samples.

Substantial improvement of microvolume UV absorption spectrometry in sensitivity, robustness and ease of operation was achieved for routine biological applications. A unique microtubing-based absorption cell (208 μm internal diameter) featuring enhanced light transmission with a liquid core waveguide technique provided dramatically enhanced absorption sensitivity, proportional to the extended path length (50 mm, from the typical 1 mm), while robust measurement performance was attained by implementation of preventive measures against bubble trapping along the light path. For plasmid DNA, absorbance at 260 nm was reliably measurable down to 0.

A High Quality Asian Genome Assembly Identifies Features of Common Missing Regions.

The current human reference genome (GRCh38), with its superior quality, has contributed significantly to genome analysis. However, GRCh38 may still underrepresent the ethnic genome, specifically for Asians, though exactly what we are missing is still elusive. Here, we juxtaposed GRCh38 with a high-contiguity genome assembly of one Korean (AK1) to show that a part of AK1 genome is missing in GRCh38 and that the missing regions harbored ~1390 putative coding elements.

A Deep Learning Approach for Estimating Traffic Density Using Data Obtained from Connected and Autonomous Probes.

The focus of this research is on the estimation of traffic density from data obtained from Connected and Autonomous Probes (CAPs). CAPs pose an advantage over expensive and invasive infrastructure such as loop detectors. CAPs maneuver their driving trajectories, sensing the presence of adjacent vehicles and distances to them by means of several electronic sensors, whose data can be used for more sophisticated traffic density estimation techniques.

Staged, Combined Management of Ruptured Vertebral Artery Dissecting Aneurysms Involving the Posterior Inferior Cerebellar Artery: Report of 4 Cases and Review of the Literature.

Background: Ruptured vertebral artery dissecting aneurysms (VADAs) involving the posterior inferior cerebellar artery (PICA) are the most difficult to treat among variations of VADAs but require prompt treatment. The major challenge is to preserve the PICA while occluding the aneurysm. Despite advances in the management of ruptured VADAs involving the PICA, each treatment, whether it is combined or not, is associated with a significant degree of risk.

Bifrontal Interhemispheric Approach Involving Cutting the Superior Sagittal Sinus for Distal Anterior Cerebral Artery Aneurysms.

Background: The unilateral interhemispheric approach for distal anterior cerebral artery aneurysms presents several risks, such as postoperative venous infarction due to occasional sacrifice of parasagittal bridging vein and postoperative frontal lobe damage due to retraction force. To overcome these risks, we used a bifrontal craniotomy with straight dural incision and cutting of the superior sagittal sinus.

Methods: We retrospectively reviewed 61 patients (42 unruptured and 19 ruptured A2 and A3 aneurysms) who under aneurysm clipping through bifrontal interhemispheric approach between March 2007 and December 2017.

Stable Isotope Labeled DNA: A New Strategy for the Quantification of Total DNA Using Liquid Chromatography-Mass Spectrometry.

Conventional DNA quantification methods require a DNA purification step that limits their reliability in estimating the original DNA amount, especially in complex matrix. To overcome this limitation, we developed a method to calibrate the variable DNA extraction efficiencies during the purification process, allowing for the accurate quantification of DNA in complex matrix. This method is based on isotope dilution-liquid chromatography-mass spectrometry using stable isotope labeled DNA (SILD) as an internal standard.

Real-Time Light-Guided Vocal Fold Injection: Ex Vivo Feasibility Study in a Canine Model.

Objective: The transcricothyroid (CT) membrane approach is a good option for office-based vocal fold injection (VFI). However, because the needle tip is invisible during injection using the CT approach, precise localization requires a high level of experience, and mastering this approach involves a steep learning curve. To overcome current limitations, we conceptualized a novel technique: real-time light-guided VFI (RL-VFI), which enables simultaneous VFI under direct visualization of the lighted needle tip.

Normalization of human RNA-seq experiments using chimpanzee RNA as a spike-in standard.

Normalization of human RNA-seq experiments employing chimpanzee RNA as a spike-in standard is reported. Human and chimpanzee RNAs exhibit single nucleotide variations (SNVs) in average 210-bp intervals. Spike-in chimpanzee RNA would behave the same as the human counterparts during the whole NGS procedures owing to the high sequence similarity.

An international comparability study on quantification of mRNA gene expression ratios: CCQM-P103.1.

Measurement of RNA can be used to study and monitor a range of infectious and non-communicable diseases, with profiling of multiple gene expression mRNA transcripts being increasingly applied to cancer stratification and prognosis. An international comparison study (Consultative Committee for Amount of Substance (CCQM)-P103.1) was performed in order to evaluate the comparability of measurements of RNA copy number ratio for multiple gene targets between two samples.

Effect of Device Rigidity and Physiological Loading on Spinal Kinematics after Dynamic Stabilization : An In-Vitro Biomechanical Study.

Objective: To investigate the effects of posterior implant rigidity on spinal kinematics at adjacent levels by utilizing a cadaveric spine model with simulated physiological loading.

Methods: Five human lumbar spinal specimens (L3 to S1) were obtained and checked for abnormalities. The fresh specimens were stripped of muscle tissue, with care taken to preserve the spinal ligaments and facet joints.

Reference Materials for Calibration of Analytical Biases in Quantification of DNA Methylation.

Most contemporary methods for the quantification of DNA methylation employ bisulfite conversion and PCR amplification. However, many reports have indicated that bisulfite-mediated PCR methodologies can result in inaccurate measurements of DNA methylation owing to amplification biases. To calibrate analytical biases in quantification of gene methylation, especially those that arise during PCR, we utilized reference materials that represent exact bisulfite-converted sequences with 0% and 100% methylation status of specific genes.

SiNG-PCRseq: Accurate inter-sequence quantification achieved by spiking-in a neighbor genome for competitive PCR amplicon sequencing.

Despite the recent technological advances in DNA quantitation by sequencing, accurate delineation of the quantitative relationship among different DNA sequences is yet to be elaborated due to difficulties in correcting the sequence-specific quantitation biases. We here developed a novel DNA quantitation method via spiking-in a neighbor genome for competitive PCR amplicon sequencing (SiNG-PCRseq). This method utilizes genome-wide chemically equivalent but easily discriminable homologous sequences with a known copy arrangement in the neighbor genome.

Determination of phosphorus impurity that directly affects quantification of microbial genomic DNA using inductively coupled plasma optical emission spectrometry.

We prepared genomic DNA from human placenta, Escherichia coli, and Bacillus subtilis using various DNA extraction methods and quantified the genomic DNA using ultraviolet (UV) spectrophotometry, capillary electrophoresis (CE), and inductively coupled plasma optical emission spectrometry (ICP-OES). Application of ICP-OES unexpectedly led to a serious overestimation of phosphorus in B. subtilis genomic DNA prepared using cetyltrimethyl ammonium bromide (CTAB).

Quantification of trace-level DNA by real-time whole genome amplification.

Quantification of trace amounts of DNA is a challenge in analytical applications where the concentration of a target DNA is very low or only limited amounts of samples are available for analysis. PCR-based methods including real-time PCR are highly sensitive and widely used for quantification of low-level DNA samples. However, ordinary PCR methods require at least one copy of a specific gene sequence for amplification and may not work for a sub-genomic amount of DNA.

Rapid and accurate determination of deoxyribonucleoside monophosphates from DNA using micellar electrokinetic chromatography with a cationic surfactant additive.

A micellar electrokinetic chromatography (MEKC) method for rapid and accurate determination of 2'-deoxyribonucleoside 5'-monophosphates (dNMPs), four structural elements of DNA, is described. MEKC separation at an optimized pH enabled complete separation of four dNMPs. The use of a cationic surfactant additive for MEKC led to the reversal of EOF, which enhanced the migration velocities of the negatively charged dNMPs.

The anti-aging gene KLOTHO is a novel target for epigenetic silencing in human cervical carcinoma.

Background: Klotho was originally characterized as an anti-aging gene that predisposed Klotho-deficient mice to a premature aging-like syndrome. Recently, KLOTHO was reported to function as a secreted Wnt antagonist and as a tumor suppressor. Epigenetic gene silencing of secreted Wnt antagonists is considered a common event in a wide range of human malignancies.

p15INK4b methylation correlates with thrombocytopenia, blast percentage, and survival in myelodysplastic syndromes in a dose dependent manner: quantitation using pyrosequencing study.

We investigated how the quantity of p15INK4b methylation related to International Prognosic Scoring System variables and survival in 74 patients with de novo myelodysplastic syndrome (MDS). Pyrosequencing of 11 consecutive CpG sites of the p15INK4b promotor region was performed, with the extent of CpG cytosine methylation assessed in terms of methylation level (MtL). Patients with >5% bone marrow blasts had higher MtL than patients with <5% blasts (10.

An international comparability study on quantification of total methyl cytosine content.

Various methods have been developed for quantitative analysis of DNA methylation. However, there is currently no reference analysis system regarding DNA methylation with which other analytical approaches can be compared and evaluated. A standard measurement system that includes reference methods and reference materials may improve comparability and credibility of data obtained from different analytical environments.

Performance evaluation of thermal cyclers for PCR in a rapid cycling condition.

The performance of thermal cyclers for polymerase chain reactions (PCR) is of great concern in terms of the reliability of PCR-based assays, particularly when rapid cycling conditions are applied to small volume reactions. In this work, the precision of the temperature controls during rapid thermal cycling was measured in 19 commercial thermal cyclers of 8 different models. The temperatures of test solutions in specific locations in each thermal block were simultaneously monitored at 1 s intervals during thermal cycling.

Well-less capillary array electrophoresis chip using hydrophilic sample bridges.

An innovative sample introduction method that facilitates extreme simplification of the layout of a capillary array electrophoresis (CAE) chip is described. Multiple samples were directly injected onto CAE channels from sample loaders using hydrophilic sample bridges, thus obviating auxiliary components of sample wells and sampling channels of a typical CAE chip. Hydrophilic sample bridges were spontaneously formed in hydrophobic surroundings to connect sample loaders to corresponding CAE channels, through which electrosample injections were effectively made.