Separation and quantification of RNA molecules using size-exclusion chromatography hyphenated with inductively coupled plasma-mass spectrometry.

The hyphenation of SEC with ICP-MS was successfully applied to RNA quantification. The developed method combines the separation technique for large biomolecules and element selective detection of ICP-MS. The separation of RNA molecules was performed under the SEC condition without additive reagents such as salts to prevent the adhesion of RNA molecules on the column resin. Fragments of RNA, which were commercially available as a ladder marker solution and certified reference materials, were successfully separated and analyzed by measuring ³¹P⁺ with this method. RNA was quantified with good repeatability (RSD of peak area; 2.7%, n = 3) and linearity (R² = 0.999) using a P standard solution as a calibrant. LOD and absolute detection limit of RNA were 6.7 µg/kg and 67 pg, respectively, which were equal to the values obtained by the analysis of a P standard solution. The accuracy of the proposed measurement was evaluated by measuring certified reference materials of RNA solutions for quantitative analysis (NMIJ CRM 6204-a). The results obtained by this method agreed with the certified values within uncertainty. The proposed analysis method, which demonstrates good accuracy and high precision and is free from interference by nucleotide analogues, qualifies as a method of quality control for the RNA synthesis and extraction process.

A coupling system of capillary gel electrophoresis with inductively coupled plasma-mass spectrometry for the determination of double stranded DNA fragments.

The coupling system of capillary gel electrophoresis (CGE) and inductively coupled plasma-mass spectrometry (ICP-MS) was newly developed and successfully applied to the double-stranded (ds) DNA quantification. The developed system combines the separation technique for large biomolecules and element selective detection of ICP-MS. This coupling was achieved by using the modified high performance concentric nebulizer (HPCN) with the PTFE tube (HPCN-PT), which can produce the liquid jet by the flow focusing effect. The HPCN-PT effectively nebulizes the highly viscous solution containing gel buffer even at a low flow rate. At a liquid flow rate of 0.010 mL min(-1) and a nebulizer gas flow rate of 1 L min(-1), the Sauter mean diameter (D3,2) of primary aerosols generated by the HPCN-PT was 3.4 µm, and over 90% (v/v) of the aerosol droplets were less than 10 µm in diameter. The electrophoresis capillary filled with gel buffer was connected to the HPCN-PT via the interface. This interface has two connectors and an electrode that can connect CE and ICP-MS. After the electrophoretic separation at atmospheric pressure, the samples were transferred to the ICP-MS through the interface by applying additional pressure. Fragments of dsDNA, which were commercially available as a ladder marker solution, were successfully separated and analyzed by measuring (31)P(+) with CGE-ICP-MS, and a linear calibration curve of the phosphorus standard solution (R(2) = 0.999) was obtained from 2.7 to 27 mg kg(-1). The detection limit (LOD) and absolute detection limit of P were 3.7 µg kg(-1) and 0.6 pg (equivalent to 6 pg of DNA), respectively. This absolute detection limit value was equal to the conventional fluorescence determination of DNA.

Development of salt-tolerance interface for an high performance liquid chromatography/inductively coupled plasma mass spectrometry system and its application to accurate quantification of DNA samples.

Accurate quantification of DNA is highly important in various fields. Determination of phosphorus by ICP-MS is one of the most effective methods for accurate quantification of DNA due to the fixed stoichiometry of phosphate to this molecule. In this paper, a smart and reliable method for accurate quantification of DNA fragments and oligodeoxythymidilic acids by hyphenated HPLC/ICP-MS equipped with a highly efficient interface device is presented. The interface was constructed of a home-made capillary-attached micronebulizer and temperature-controllable cyclonic spray chamber (IsoMist). As a separation column for DNA samples, home-made methacrylate-based weak anion-exchange monolith was employed. Some parameters, which include composition of mobile phase, gradient program, inner and outer diameters of capillary, temperature of spray chamber etc., were optimized to find the best performance for separation and accurate quantification of DNA samples. The proposed system could achieve many advantages, such as total consumption for small amount sample analysis, salt-tolerance for hyphenated analysis, high accuracy and precision for quantitative analysis. Using this proposed system, the samples of 20 bp DNA ladder (20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 300, 400, 500 base pairs) and oligodeoxythymidilic acids (dT(12-18)) were rapidly separated and accurately quantified.

Quantification of phosphorus in DNA using capillary electrophoresis hyphenated with inductively coupled plasma mass spectrometry.

We have analyzed phosphorus in an enzymatically digested DNA molecule using capillary electrophoresis (CE) hyphenated with inductively coupled plasma mass spectrometry (ICP-MS). The DNA concentration was quantified by the phosphorus value obtained in the CE-ICP-MS analysis. The CE-ICP-MS measurement, for which the interface device AIF-01 equipped three layered nebulizer was adopted, was achieved with limited µL/min nebulizing without loss of sample in the vaporizing chamber. The samples of nucleotides and free phosphate were separated well in the CE-ICP-MS measurement, and the calibration curve (0.1-10µg/mL) of the phosphorus showed a linear (R(2)=0.999) increase in intensity. After digestion of the 100-bp double-strand DNA sample to deoxyribonucleotide-5'-monophosphates (dNMPs) by phosphodiesterase-I, phosphorus was detected by CE-ICP-MS without further purification steps. In this study, we applied two calculation schemes of DNA analysis using a dNMP concentration obtained from CE-ICP-MS. Comparative CE-ICP-MS analysis with DNA digested to dNMPs showed that the assay gave an equal value obtained from the total DNA quantification using fluorescence detection. The detection limits of the DNA sample obtained from these species and phosphorus in nucleotides using CE-ICP-MS were 3.1-26ng/mL. These LOD values were equal to the conventional fluorescence determination of DNA.

Generation of functional NKT cells in vitro from embryonic stem cells bearing rearranged invariant Valpha14-Jalpha18 TCRalpha gene.

Establishment of a system with efficient generation of natural killer T (NKT) cells from embryonic stem (ES) cells would enable us to identify the cells with NKT-cell potential and obtain NKT cells with desired function. Here, using cloned ES (NKT-ES) cells generated by the transfer of nuclei from mature NKT cells, we have established a culture system that preferentially developed functional NKT cells and also identified early NKT progenitors, which first appeared on day 11 as a c-kit(+) population in the cocultures on OP9 cells with expression of Notch ligand, delta-like1 (OP9/Dll-1) and became c-kit(lo/-) on day 14. Interestingly, in the presence of Notch signals, NKT-ES cells differentiated only to thymic CD44(lo) CD24(hi) NKT cells producing mainly interleukin-4 (IL-4), whereas NKT cells resembling CD44(hi) CD24(lo) liver NKT cells producing mainly interferon gamma (IFN-gamma) and exhibiting strong adjuvant activity in vivo were developed in the switch culture starting at day 14 in the absence of Notch. The cloned ES culture system offers a new opportunity for the elucidation of the molecular events on NKT-cell development and for the establishment of NKT-cell therapy.

Determination of phosphorus using capillary electrophoresis and micro-high-performance liquid chromatography hyphenated with inductively coupled plasma mass spectrometry for the quantification of nucleotides.

We performed the quantification of phosphorus in deoxynucleotides using capillary electrophoresis (CE) and micro-HPLC (microHPLC) hyphenated with inductively coupled plasma mass spectrometry (ICP-MS). DNA and its component units have conventionally been determined by photometry; however, more selective and sensitive methods are needed for small biological samples. CE and microHPLC offer the advantages of good separation and small consumption of samples, and ICP-MS is a highly sensitive technique for the determination of a chemical element. Therefore, we have developed an interface device for combining CE and microHPLC with ICP-MS for quantifying nucleotides based on phosphorus content. The interface utilizes 4.5 microL/min for nebulizing and effective introduction of the sample into ICP. The samples of nucleotides and free phosphoric acid were well separated in the CE-ICP-MS measurement, and the calibration curves (1-100 microg/mL) of the nucleotides showed a linear (R2>0.999) increase in intensity. Similarly, the samples of nucleotides were baseline separated using microHPLC-ICP-MS, and the calibration curves of the nucleotides were linear (R2>0.998). The detection limits of these species and phosphorus in nucleotides using CE-ICP-MS and microHPLC-ICP-MS were 0.77-6.5 ng/mL and 4.0-6.5 ng/mL, respectively. These values were about one or two orders lower than those in a previous report. The sample volumes of these experiments were calculated to be about 10 nL and 50 nL per analysis. Therefore, these analytical methods have the potential to be useful for the determination of biological samples, such as DNA and RNA molecules.

Quantification of oligonucleotide containing sequence failure product: comparison of isotope dilution mass spectrometry with other quantification methods.

There is an increasing demand to develop a method for accurate quantification of DNA. Because current methods such as the ultraviolet (UV) absorption-based method are only capable of relative quantification, the quantification result depends completely on the reference material. To achieve accurate quantification of DNA, we have performed isotope dilution mass spectrometry (ID MS)-based quantification of oligonucleotides. We chose a 20-mer synthetic oligonucleotide as the analyte with a longer sequence failure product. Oligonucleotides sometimes contain sequence failure products, which are difficult to remove. It is important to quantify a target product in such mixture. After evaluating the content of the sequence failure product, the analyte spiked with stable isotopically labeled deoxynucleotide monophosphates (dNMPs) was digested by enzyme to its constituent dNMPs or deoxynucleosides, and quantified by liquid chromatography-mass spectrometry. The obtained mass fractions of the 20-mer oligonucleotide showed a good agreement with the results based on phosphate analysis by inductively coupled plasma-optical emission spectrometry and ion chromatography. UV absorption, the general method for DNA quantification, resulted in underestimation. On the other hand, the mass fraction obtained by the gravimetric method was overestimated. This study shows that the ID MS method can determine the precise mass fraction of the target oligonucleotide with the sequence failure product and possesses potential as the primary method for the certification of DNA as a reference material.

An adjuvant role of in situ dendritic cells (DCs) in linking innate and adaptive immunity.

Dendritic cells (DCs) work as a natural adjuvant to elicit T cell immunity. Though DCs have been widely used in immunotherapy, little is known about their number and function in patients with cancer or autoimmune disease. In recent studies, antigen has been targeted to DCs through DC-specific receptors, such as DEC205, the mannose receptor and dying cell receptors. However, antigen captured by DCs in the absence of danger signals induces tolerance. Therefore, the duration and/or magnitude of danger signals plays a crucial role in generating an immunogeneic response. Various danger signals, i.e., pathogen-associated molecular pattern (PAMP), damage-associated molecular pattern (DAMP) and the activation of innate lymphocytes, serve as maturation signals for DCs. An immunotherapeutic approach which delivers both maturation signals and antigen to DCs would link the innate and adaptive arms of the immune system for a more effective and global immune response. It is therefore crucial to determine optimal conditions for antigen delivery to DCs in an environment suited to maximally stimulate the immune system.