Makiko Ichikawa

Identification of AhR-regulated genes involved in PAH-induced immunotoxicity using a highly-sensitive DNA chip, 3D-GeneTM Human Immunity and Metabolic Syndrome 9k

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants with various toxic effects including immune suppression. However, the molecular mechanism of their toxicity has not been fully clarified. The purpose of this study was to identify novel aryl hydrocarbon receptor (AhR)-regulated genes involved in PAH-induced immunotoxicity using a highly-sensitive DNA chip, 3D-Gene(TM) Human Immunity & Metabolic Syndrome 9k. Leucine-rich repeat-containing protein 25, glucosaminyl (N-acetyl) transferase 3 (GCNT3), thyroxine-binding globulin, aldehyde dehydrogenase 8A1, diacylglycerol O-acyltransferase homolog 2 (DGAT2), haptoglobin, neuron navigator 2 isoform 1, hemopexin and bile acid receptor were found to be up- or down-regulated by PAHs via AhR. Among these genes, GCTN3 and DGAT2 were responsible for immune responses. Therefore, disruption of the expression of these genes via AhR may be one of the causes of the immunotoxicity of PAHs.

A Combination of Extraction Reagent and DNA Microarray That Allows for the Detection of Global MiRNA Profiles from Serum/Plasma

In recent years, miRNAs have been shown to exist stably in serum (plasma) and have drawn attention particularly as markers for diagnosis of diseases, evaluation of therapeutic effects, selection of treatment in clinical studies, and others.However, RNAs in serum (plasma) exist only in low amounts (0.1-1 ng/mL), and analysis with reproducibility is difficult. Therefore, we have developed a combination of an extraction reagent and a unique miRNA screening platform which allows for the rapid analysis and high-throughput detection of alterations in miRNA levels in serum/plasma samples. This offers the potential for the identification of novel biomarkers to specific diseases or conditions which may inform upon future diagnostic approaches. The features of this prescription include (1) an extraction method that can obtain high-purity RNA (high reproducibility and stability), (2) a straightforward, easy-to-use extraction procedure a simple method without complicated extraction operations, and (3) increased number of detected genes and data reproducibility using high sensitivity DNA chips.

Time-saving multiplex detection of single nucleotide polymorphisms by ultrasensitive DNA microarray

Rapid and multiplex detection system using an ultrasensitive DNA microarray was developed and utilized for the analysis of six pharmacokinetically relevant single nucleotide polymorphisms (SNPs) (MDR1-C1236T, MDR1-G2677TA, MDR1-C3435T, CYP3A5-A6986G, CYP2C19-G681A, CYP2C19-G636A) from blood samples derived from liver transplant patients. The SNP detection system is comprised of three processes: multiplex PCR, single base extension with fluorescently labelled di-deoxy-nucleotides and detection by DNA microarray. The entire workflow of this system completes within 5 h. The final genotype call was obtained statistically by Mahalanobis distance which was calculated from the bi-coloured fluorescent signals detected by the microarray. In order to detect the six SNPs, this system required only 50 copies of genomic DNA, and the obtained detection calls completely matched with the results by the sequencing-based genotyping method. With the high sensitivity and rapid processing, our SNP detection system utilizing ultrasensitive microarray is a promising device applicable for diagnostic utility.

Abstract 5034: Healthy and cancerous serum RNA profiling by the novel RNA extraction reagent and highly sensitive DNA chip

Proteins, metabolites and DNA are already known as components of serum or plasma biomarkers, however RNA has not been a strong biomarker candidate because of its instability. Exosomes that are small vesicles secreted by various cells are recently reported to play important roles in intercellular communications by transferring proteins, DNA and also RNA to distant cells through circulatory system. Surprisingly, the exosomal RNA in serum preserves its integrity and thus holds a potential to be a new blood biomarkers. In this report, we show the exhaustive analysis of miRNA and mRNA in serum by DNA chip for the highly purified RNA extracted with a novel reagent. Serum contains various types of nucleic acids, mainly small RNA, mRNA, and also short DNA fragment. We suppose that the contamination of DNA to the extracted RNA often causes a discrepancy between the DNA chip analysis and qRT-PCR validation. The novel reagent was able to extract RNA from serum without contamination of short DNA fragment, resulting in better RNA quantification and decreasing DNA-related noise outputs. Using this novel RNA extraction reagent and the highly sensitive DNA chip “3D-Gene,” we analyzed healthy and cancerous serum miRNA profiles. Over 500 miRNAs are detected in healthy and cancerous sera reproducibly, and some miRNAs were detected specifically in cancerous sera, such as breast, gastric, cervix cancer. In addition, we detected over 19,000 mRNAs from amplified RNA which were similarly extracted from healthy serum and detected by the DNA chip. This indicates that not only serum miRNA but also serum mRNA have a potential capability to be a biomarker. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5034. doi:1538-7445.AM2012-5034