Reliable biological and multi-omics research through biometrology.

Metrology is the science of measurement and its applications, whereas biometrology is the science of biological measurement and its applications. Biometrology aims to achieve accuracy and consistency of biological measurements by focusing on the development of metrological traceability, biological reference measurement procedures, and reference materials. Irreproducibility of biological and multi-omics research results from different laboratories, platforms, and analysis methods is hampering the translation of research into clinical uses and can often be attributed to the lack of biologists' attention to the general principles of metrology. In this paper, the progresses of biometrology including metrology on nucleic acid, protein, and cell measurements and its impacts on the improvement of reliability and comparability in biological research are reviewed. Challenges in obtaining more reliable biological and multi-omics measurements due to the lack of primary reference measurement procedures and new standards for biological reference materials faced by biometrology are discussed. In the future, in addition to establishing reliable reference measurement procedures, developing reference materials from single or multiple parameters to multi-omics scale should be emphasized. Thinking in way of biometrology is warranted for facilitating the translation of high-throughput omics research into clinical practices.

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. Tagged RNAs were exhaustively counted one by one using a high-sensitivity capillary-based flow cytometric setup. MS2 viral RNA was quantified as a model RNA for which essential parameters, including probe numbers, probe concentration, and hybridization conditions, were optimized for the best performance. Using 70 oligonucleotide probes, MS2 RNA was quantified with 2.0% relative standard deviation, and its validity was assessed by comparison with other RNA quantification methods such as droplet digital PCR and UV spectrophotometry. The observed comparability indicated that the proposed method is unlikely to have a substantial bias. It works for a substantially lower-level RNA than UV and avoids the potential variability in the yield of the RT reaction of RT-qPCR. Therefore, the proposed method could be a valuable addition to current methods and could be further developed as a standard reference method for RNA quantification.

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 pBR322 plasmid DNA, absorbance at 260 nm was reliably measurable down to 0.1 ng/µl with repeatability typically 2-3% RSD. The detection limit was 0.03 ng/µl dsDNA, far lower than the current state-of-the-art âˆ¼1 ng/µl. Sample consumption for each measurement was 2.4 µl. Automated operation implemented for the first time in microvolume spectrophotometry facilitated the ease in handling with small-volume biological samples.

Assessment of Digital PCR as a Primary Reference Measurement Procedure to Support Advances in Precision Medicine.

BACKGROUND: Genetic testing of tumor tissue and circulating cell-free DNA for somatic variants guides patient treatment of many cancers. Such measurements will be fundamental in the future support of precision medicine. However, there are currently no primary reference measurement procedures available for nucleic acid quantification that would support translation of tests for circulating tumor DNA into routine use. METHODS: We assessed the accuracy of digital PCR (dPCR) for copy number quantification of a frequently occurring single-nucleotide variant in colorectal cancer (KRAS c.35G>A, p.Gly12Asp, from hereon termed G12D) by evaluating potential sources of uncertainty that influence dPCR measurement. RESULTS: Concentration values for samples of KRAS G12D and wild-type plasmid templates varied by <1.2-fold when measured using 5 different assays with varying detection chemistry (hydrolysis, scorpion probes, and intercalating dyes) and <1.3-fold with 4 commercial dPCR platforms. Measurement trueness of a selected dPCR assay and platform was validated by comparison with an orthogonal method (inductively coupled plasma mass spectrometry). The candidate dPCR reference measurement procedure showed linear quantification over a wide range of copies per reaction and high repeatability and interlaboratory reproducibility (CV, 2%-8% and 5%-10%, respectively). CONCLUSIONS: This work validates dPCR as an SI-traceable reference measurement procedure based on enumeration and demonstrates how it can be applied for assignment of copy number concentration and fractional abundance values to DNA reference materials in an aqueous solution. High-accuracy measurements using dPCR will support the implementation and traceable standardization of molecular diagnostic procedures needed for advancements in precision medicine.

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. After discrimination of species origins of the NGS reads based on SNVs, the chimpanzee reads were used to read-by-read normalize biases and variations of human reads. By this approach, as many as 10,119 transcripts were simultaneously normalized for the entire NGS procedures leading to accurate and reproducible quantification of differential gene expression. In addition, incomparable data sets from different in-process degradations or from different library preparation methods were made well comparable by the normalization. Based on these results, we expect that the normalization approaches using near neighbor genomes as internal standards could be employed as a standard protocol, which will improve both accuracy and comparability of NGS results across different sample batches, laboratories and NGS platforms.

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. Six exogenous synthetic targets comprising of External RNA Control Consortium (ERCC) standards were measured alongside transcripts for three endogenous gene targets present in the background of human cell line RNA. The study was carried out under the auspices of the Nucleic Acids (formerly Bioanalysis) Working Group of the CCQM. It was coordinated by LGC (United Kingdom) with the support of National Institute of Standards and Technology (USA) and results were submitted from thirteen National Metrology Institutes and Designated Institutes. The majority of laboratories performed RNA measurements using RT-qPCR, with datasets also being submitted by two laboratories based on reverse transcription digital polymerase chain reaction and one laboratory using a next-generation sequencing method. In RT-qPCR analysis, the RNA copy number ratios between the two samples were quantified using either a standard curve or a relative quantification approach. In general, good agreement was observed between the reported results of ERCC RNA copy number ratio measurements. Measurements of the RNA copy number ratios for endogenous genes between the two samples were also consistent between the majority of laboratories. Some differences in the reported values and confidence intervals ('measurement uncertainties') were noted which may be attributable to choice of measurement method or quantification approach. This highlights the need for standardised practices for the calculation of fold change ratios and uncertainties in the area of gene expression profiling.

Accurate quantification of supercoiled DNA by digital PCR.

Digital PCR (dPCR) as an enumeration-based quantification method is capable of quantifying the DNA copy number without the help of standards. However, it can generate false results when the PCR conditions are not optimized. A recent international comparison (CCQM P154) showed that most laboratories significantly underestimated the concentration of supercoiled plasmid DNA by dPCR. Mostly, supercoiled DNAs are linearized before dPCR to avoid such underestimations. The present study was conducted to overcome this problem. In the bilateral comparison, the National Institute of Metrology, China (NIM) optimized and applied dPCR for supercoiled DNA determination, whereas Korea Research Institute of Standards and Science (KRISS) prepared the unknown samples and quantified them by flow cytometry. In this study, several factors like selection of the PCR master mix, the fluorescent label, and the position of the primers were evaluated for quantifying supercoiled DNA by dPCR. This work confirmed that a 16S PCR master mix avoided poor amplification of the supercoiled DNA, whereas HEX labels on dPCR probe resulted in robust amplification curves. Optimizing the dPCR assay based on these two observations resulted in accurate quantification of supercoiled DNA without preanalytical linearization. This result was validated in close agreement (101~113%) with the result from flow cytometry.

International Comparison of Enumeration-Based Quantification of DNA Copy-Concentration Using Flow Cytometric Counting and Digital Polymerase Chain Reaction.

Enumeration-based determination of DNA copy-concentration was assessed through an international comparison among national metrology institutes (NMIs) and designated institutes (DIs). Enumeration-based quantification does not require a calibration standard thereby providing a route to "absolute quantification", which offers the potential for reliable value assignments of DNA reference materials, and International System of Units (SI) traceability to copy number 1 through accurate counting. In this study, 2 enumeration-based methods, flow cytometric (FCM) counting and the digital polymerase chain reaction (dPCR), were compared to quantify a solution of the pBR322 plasmid at a concentration of several thousand copies per microliter. In addition, 2 orthogonal chemical-analysis methods based on nucleotide quantification, isotope-dilution mass spectrometry (IDMS) and capillary electrophoresis (CE) were applied to quantify a more concentrated solution of the plasmid. Although 9 dPCR results from 8 laboratories showed some dispersion (relative standard deviation [RSD] = 11.8%), their means were closely aligned with those of the FCM-based counting method and the orthogonal chemical-analysis methods, corrected for gravimetric dilution factors. Using the means of dPCR results, the RSD of all 4 methods was 1.8%, which strongly supported the validity of the recent enumeration approaches. Despite a good overall agreement, the individual dPCR results were not sufficiently covered by the reported measurement uncertainties. These findings suggest that some laboratories may not have considered all factors contributing to the measurement uncertainty of dPCR, and further investigation of this possibility is warranted.

Development and co-validation of porcine insulin certified reference material by high-performance liquid chromatography-isotope dilution mass spectrometry.

This article concerns the development and co-validation of a porcine insulin (pINS) certified reference material (CRM) produced by the National Institute of Metrology, People's Republic of China. Each CRM unit contained about 15 mg of purified solid pINS. The moisture content, amount of ignition residue, molecular mass, and purity of the pINS were measured. Both high-performance liquid chromatography-isotope dilution mass spectrometry and a purity deduction method were used to determine the mass fraction of the pINS. Fifteen units were selected to study the between-bottle homogeneity, and no inhomogeneity was observed. A stability study concluded that the CRM was stable for at least 12 months at -20 °C. The certified value of the CRM was (0.892 ± 0.036) g/g. A co-validation of the CRM was performed among Chinese, Japanese, and Korean laboratories under the framework of the Asian Collaboration on Reference Materials. The co-validation results agreed well with the certified value of the CRM. Consequently, the pINS CRM may be used as a calibration material or as a validation standard for pharmaceutical purposes to improve the quality of pharmaceutical products.

Quantification of recombinant human erythropoietin by amino acid analysis using isotope dilution liquid chromatography-tandem mass spectrometry.

Herein, we describe an accurate method for protein quantification based on conventional acid hydrolysis and an isotope dilution-ultra performance liquid chromatography-tandem mass spectrometry method. The analyte protein, recombinant human erythropoietin (rhEPO), was effectively hydrolyzed by incubation with 8 mol/L hydrochloric acid at 130 °C for 48 h, in which at least 1 µmol/kg of rhEPO was treated to avoid possible degradation of released amino acids during hydrolysis. Prior to hydrolysis, sample solution was subjected to ultrafiltration to eliminate potential interfering substances. In a reversed-phase column, the analytes (phenylalanine, proline, and valine) were separated within 3 min using gradient elution comprising 20 % (v/v) acetonitrile and 10 mmol/L ammonium acetate, both containing 0.3 % (v/v) trifluoroacetic acid. The optimized hydrolysis and analytical conditions in our study were strictly validated in terms of accuracy and precision, and were suitable for the accurate quantification of rhEPO. Certified rhEPO was analyzed using a conventional biochemical assay kit as an additional working calibrant for the quantification of EPO and improved the accuracy. The optimized protocol is suitable for the accurate quantification of rhEPO and satisfactorily serves as a reference analytical procedure for the certification of rhEPO and similar proteins.

Amino acid analysis of dried blood spots for diagnosis of phenylketonuria using capillary electrophoresis-mass spectrometry equipped with a sheathless electrospray ionization interface.

We describe a capillary electrophoresis-mass spectrometry (CE-MS) method for newborn screening of a representative amino acid metabolic disease, namely, phenylketonuria (PKU). Underivatized phenylalanine and tyrosine in a dried blood spot (DBS) were simultaneously determined by CE-MS equipped with an ionophore membrane-packed sheathless electrospray ionization interface, which was developed by our group. The method was optimized for rapid determination of the underivatized amino acids, phenylalanine and tyrosine extracted from a DBS. Under the optimized conditions, the limit of detection of phenylalanine and tyrosine (signal-to-noise ratio, 3) was 0.03 and 0.07 mg/L in DBS, respectively, with a CE run time of less than 3 min. For repeated runs of a sample, coefficients of variation (CVs) for migration time were less than 3.7%, whereas CVs for the area ratio under the curve were 2.1 and 2.9% for 20 consecutive runs of 49.5 mg/kg Phe and 36.2 mg/kg Tyr, respectively. However, the relative standard deviations of intra- and interday assays for DBS samples were <6.2 and <5.8%, respectively, which were substantially due to sample extraction from DBS. The analytical method was applied to real clinical samples of Korean neonates, and results were compared with those of conventional methods for PKU diagnosis, which required reference analytical methods such as isotope dilution CE-MS or high-performance liquid chromatography-mass spectrometry for quality assurance of the conventional kit-based assays. The distinct advantages of high sensitivity and extremely low sample volume, as well as a simple, easy, and economic sample pretreatment, were demonstrated for the proposed method.

Development of an online microbore hollow fiber enzyme reactor coupled with nanoflow liquid chromatography-tandem mass spectrometry for global proteomics.

In this study, we report the development of a microbore hollow fiber enzyme reactor (mHFER) coupled to nanoflow liquid chromatography-tandem mass spectrometry (nLC-ESI-MS/MS) for the online digestion or selective enrichment of glycopeptides and analysis of proteins. With mHFER, enzymatic digestion of protein could be achieved by continuous flow within a very small volume (~10 µL) of mHF inserted in a PEEK tube. Digested peptides exited through the pores of the hollow fiber membrane wall to external single or multiplexed trap columns for nLC-ESI-MS/MS analysis. Evaluation of online mHFER-nLC-ESI-MS/MS system was made with bovine serum albumin (BSA) by varying the temperature of digestion and the amount of protein injected. We evaluated the ability of the mHFER system to enrich glycopeptides by injecting a mixture of lectin (concanavalin A) and digested peptides from α-1-acid glycoprotein (AGP) into the mHFER, followed by delivery of PNGase F for endoglycosidic digestion. Nonglycosylated peptides unbound to lectins eluted at the first breakthrough run while N-linked glycopeptides eluted after the endoglycosidic digestion. The developed method was applied to urine samples from patients with prostate cancer and controls; 67 N-linked glycopeptides were identified and relative differences in glycopeptide content between patient and control samples were determined.

Capillary electrophoresis mass spectrometry with sheathless electrospray ionization for high sensitivity analysis of underivatized amino acids.

A high durability sheathless electrospray ionization interface of CE-MS is applied for the sensitive analysis of underivatized amino acids. The sheathless interface was realized using an ionophore membrane-packed electro-conduction channel. The interface functioned well with a volatile alkaline background electrolyte (BGE) and uncoated fused-silica capillaries for CE-MS analysis of underivatized amino acids. High electroosmotic flow with alkaline BGE facilitated high separation efficiency (>100,000 theoretical plates) and short analysis time (<15 min). Both the short-term stability and long-term durability are particularly suited for routine applications. Using electrokinetic injection and the multiple reaction monitoring (MRM) mode with a triple-quadrupole analyzer, high sensitivity was achieved, which yielded detection limits of 0.05-0.81 µM. For the quantitation of underivatized amino acids, quantification precisions (RSDs) for intra- and inter-day analyses were less than 3%. Recoveries from serum were 96.3-101.8% for isotope dilution mass spectrometry (IDMS). When compared with HPLC-IDMS for human serum samples, highly agreeable (96.9-102.0%) results were obtained with the proposed CE-IDMS method.

Spectrofluorimetric study of the interaction between europium(III) and moxifloxacin in micellar solution and its analytical application.

A sensitive spectrofluorimetric method has been developed for the determination of moxifloxacin (MOX) using europium(III)-MOX complex as a fluorescence probe in the presence of an anionic surfactant, sodium dodecyl benzene sulfonate (SDBS). The fluorescence (FL) intensity of Eu(3+) was enhanced by complexation with MOX at 614 nm after excitation at 373 nm. The FL intensity of the Eu(3+)-MOX complex was significantly intensified in the presence of SDBS. Under the optimum conditions, it was found that the enhanced FL intensity of the system showed a good linear relationship with the concentration of MOX over the range of 1.8 × 10(-11)-7.3 × 10(-9) g mL(-1) with a correlation coefficient of 0.9998. The limit of detection of MOX was found to be 2.8 × 10(-12) g mL(-1) with relative standard deviation (RSD) of 1.25% for 5 replicate determination of 1.5 × 10(-8) g mL(-1) MOX. The proposed method is simple, offers higher sensitivity with wide linear range and can be successfully applied to determine MOX in pharmaceutical and biological samples with good reproducibility. The luminescence mechanism is also discussed in detail with ultraviolet absorption spectra.

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. We suggest a real-time whole genome amplification method adopting the degenerate oligonucleotide primed PCR (DOP-PCR) for quantification of sub-genomic amounts of DNA. This approach enabled quantification of sub-picogram amounts of DNA independently of their sequences. When the method was applied to the human placental DNA of which amount was accurately determined by inductively coupled plasma-optical emission spectroscopy (ICP-OES), an accurate and stable quantification capability for DNA samples ranging from 80 fg to 8 ng was obtained. In blind tests of laboratory-prepared DNA samples, measurement accuracies of 7.4%, -2.1%, and -13.9% with analytical precisions around 15% were achieved for 400-pg, 4-pg, and 400-fg DNA samples, respectively. A similar quantification capability was also observed for other DNA species from calf, E. coli, and lambda phage. Therefore, when provided with an appropriate standard DNA, the suggested real-time DOP-PCR method can be used as a universal method for quantification of trace amounts of DNA.

Quantification of human growth hormone by amino acid composition analysis using isotope dilution liquid-chromatography tandem mass spectrometry.

We describe an accurate method for protein quantification based on conventional acid hydrolysis and an isotope dilution-HPLC-mass spectrometry (ID-HPLC-MS) method. Sample purity was confirmed using capillary zone electrophoresis, HPLC and MS. The analyte protein, human growth hormone (hGH), was effectively hydrolyzed by incubation with 8 M hydrochloric acid at 130 °C for 48 h, where at least 1 µM of hGH was treated to avoid possible degradation of released amino acids during hydrolysis. Using a reversed-phase column, the analytes (isoleucine, phenylalanine, proline and valine) were separated within 5 min using an isocratic eluent comprising 10% acetonitrile containing 0.1% trifluoroacetic acid. The detection limit (signal to noise ratio of 3) of amino acids was 5.5-6.2 fmol per injection. The quantification precision (RSD) of amino acids for intra- and inter-day assays was less than 0.98% and 0.39%, respectively. Comparison with other biochemical and instrumental methods revealed substantially higher accuracy and reproducibility of the ID-HPLC-MS/MS method as expected. The optimized hydrolysis and analytical conditions in our study were suitable for accurate quantification of hGH.

Crystal structure of prephenate dehydrogenase from Streptococcus mutans.

Prephenate dehydrogenase (PDH) is a bacterial enzyme that catalyzes conversion of prephenate to 4-hydroxyphenylpyruvate through the oxidative decarboxylation pathway for tyrosine biosynthesis. This enzymatic pathway exists in prokaryotes but is absent in mammals, indicating that it is a potential target for the development of new antibiotics. The crystal structure of PDH from Streptococcus mutans in a complex with NAD(+) shows that the enzyme exists as a homo-dimer, each monomer consisting of two domains, a modified nucleotide binding N-terminal domain and a helical prephenate C-terminal binding domain. The latter is the dimerization domain. A structural comparison of PDHs from mesophilic S. mutans and thermophilic Aquifex aeolicus showed differences in the long loop between ß6 and ß7, which may be a reason for the high K(m) values of PDH from Streptococcus mutans.

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. Under the optimized condition, full-baseline separation of the four dNMPs assuring accurate peak integration was obtained within 5 min. For the given separation condition, pH-mediated on-column sample stacking was optimized and applied to enhance sensitivity up to 6-fold. Analytical precision was improved by spiking iothalamate as an internal standard. The accuracy of dNMP quantitation was ensured with dNMP standard solutions determined by inductively coupled plasma-optical emission spectroscopy that measured phosphorous quantity. Performance of the proposed method was ultimately proven by accurate quantitation of a DNA oligonucleotide that was enzymatically hydrolyzed prior to dNMP analysis. The proposed MEKC method turned out to be a reliable analytical method for dNMPs that features high speed, high sensitivity, and high precision, and could be utilized for high-accuracy determination of the amount of DNA as well as the base composition of DNA.

A sheathless CE/ESI-MS interface with an ionophore membrane-packed electro-conduction channel.

A robust and convenient sheathless CE/ESI-MS interface realized with an ionophore membrane-packed electro-conduction channel is described. Sheathless interfaces that may provide higher sensitivity for MS detection than sheath flow-supported interfaces generally show instability and short lifetimes due to their imperfection in making an electrical contact with the emitter tip. In this work, we designed a sheathless interface based on an ionophore membrane-packed electro-conduction channel. At the joining point of the CE capillary and the emitter capillary, the conduction channel was implemented toward the exterior of the interface body, where a platinum wire electrode was placed. The conduction channel transferred the electric field from the external Pt electrode to the joining point, but prevented the effluent of CE from leaking. The interface body was designed to have receptacles for standard capillary tubing with finger-tight fittings, which allowed easy replacement of capillary tubing. Stable electrospray was observed for an extended time period without any signs of bubbling or damage to the emitter tip. No significant increment of dead-volume at the interface was observed for well-aligned capillaries. Sensitive and stable CE-MS detection of the model compound of creatinine and uric acid was demonstrated.

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. In an effort to establish a standard system for measurement of DNA methylation, the Korea Research Institute of Standards and Science (KRISS) coordinated an international comparison study among different national metrology institutes. An initial stage of the study involved an intercomparison regarding quantitative measurement of total methyl cytosine contents in artificially constructed DNA samples. The measurement principle involved measurement of dNMP contents following enzymatic hydrolysis of DNA samples. Results of the study showed good comparability among four of five participants and close agreement with reference values assigned by the coordinating laboratory. Conflicting data from one participant may have resulted from incomplete hydrolysis of samples due to use of insufficient amounts of enzymes. These results indicate that comparable and accurate results can be obtained from different measurement environments if digestion conditions are controlled appropriately and valid calibration systems are employed.