Tomoharu Kajiyama

Quantitative analysis of gene expression in a single cell by qPCR

We developed a quantitative PCR method featuring a reusable single-cell cDNA library immobilized on beads for measuring the expression of multiple genes in a single cell. We used this method to analyze multiple cDNA targets (from several copies to several hundred thousand copies) with an experimental error of 15.9% or less. This method is sufficiently accurate to investigate the heterogeneity of single cells.

Multiplex SNP typing by bioluminometric assay coupled with terminator incorporation

A multiplex single-nucleotide polymorphism (SNP) typing platform using ‘bioluminometric assay coupled with terminator [2′,3′-dideoxynucleoside triphosphates (ddNTPs)] incorporation’ (named ‘BATI’ for short) was developed. All of the reactions are carried out in a single reaction chamber containing target DNAs, DNA polymerase, reagents necessary for converting PPi into ATP and reagents for luciferase reaction. Each of the four ddNTPs is dispensed into the reaction chamber in turn. PPi is released by a nucleotide incorporation reaction and is used to produce ATP when the ddNTP dispensed is complementary to the base in a template. The ATP is used in a luciferase reaction to release visible light. Only 1 nt is incorporated into a template at a time because ddNTPs do not have a 3′ hydroxyl group. This feature greatly simplifies a sequencing spectrum. The luminescence is proportional to the amount of template incorporated. Only one peak appears in the spectrum of a homozygote sample, and two peaks at the same intensity appear for a heterozygote sample. In comparison with pyrosequencing using dNTP, the spectrum obtained by BATI is very simple, and it is very easy to determine SNPs accurately from it. As only one base is extended at a time and the extension signals are quantitative, the observed spectrum pattern is uniquely determined even for a sample containing multiplex SNPs. We have successfully used BATI to type various samples containing plural target sequence areas. The measurements can be carried out with an inexpensive and small luminometer using a photodiode array as the detector. It takes only a few minutes to determine multiplex SNPs. These results indicate that this novel multiplexed approach can significantly decrease the cost of SNP typing and increase the typing throughput with an inexpensive and small luminometer.

Spatial Regulation of the Gene Expression Response to Shade in Arabidopsis Seedlings

The shade avoidance response, which allows plants to escape from nearby competitors, is triggered by a reduction in the PFR form of phytochrome in response to shade. Classic physiological experiments have demonstrated that the shade signal perceived by the leaves is transmitted to the other parts of the plant. Recently, a simple method was developed to analyze the transcriptome in a single microgram tissue sample. In the present study, we adopted this method to conduct organ-specific transcriptomic analysis of the shade avoidance response in Arabidopsis seedlings. The shoot apical samples, which contained the meristem, basal parts of leaf primordia and short fragments of vasculature, were collected from the topmost part of the hypocotyl and subjected to RNA sequencing analysis. Unexpectedly, many more genes were up-regulated in the shoot apical region than in the cotyledons. Spotlight irradiation demonstrated that the apex-responsive genes were mainly controlled by phytochrome in the cotyledons. In accordance with the involvement of many auxin-responsive genes in this category, auxin biosynthesis was genetically shown to be essential for this response. In contrast, organ-autonomous regulation was more important for the genes that were up-regulated preferentially either in the cotyledons or in both the cotyledons and the apical region. Their responses to shade depended variously on auxin and PIFs (phytochrome-interacting factors), indicating the mechanistic diversity of the organ-autonomous response. Finally, we examined the expression of the auxin synthesis genes, the YUC genes, and found that three YUC genes, which were differently spatially regulated, co-ordinately elevated the auxin level within the shoot apical region.

Sensitive and specific colorimetric DNA detection by invasive reaction coupled with nicking endonuclease-assisted nanoparticles amplification

Colorimetric DNA detection is preferable to methods in clinical molecular diagnostics, because no expensive equipment is required. Although many gold nanoparticle-based colorimetric DNA detection strategies have been developed to analyze DNA sequences of interest, few of them can detect somatic mutations due to their insufficient specificity. In this study, we proposed a colorimetric DNA detection method by coupling invasive reaction with nicking endonuclease-assisted nanoparticles amplification (IR-NEANA). A target DNA firstly produces many flaps by invasive reaction. Then the flaps are converted to targets of nicking reaction-assisted nanoparticles amplification by ligation reaction to produce the color change of AuNPs, which can be observed by naked eyes. The detection limit of IR-NEANA was determined as 1pM. Most importantly, the specificity of the method is high enough to pick up as low as 1% mutant from a large amount of wild-type DNA backgrounds. The EGFR gene mutated at c.2573 T>G in 9 tissue samples from non-small cell lung cancer patients were successfully detected by using IR-NEANA, suggesting that our proposed method can be used to detect somatic mutations in biological samples.

Digital detection of multiple minority mutants in stool DNA for noninvasive colorectal cancer diagnosis.

Somatic mutations in stool DNA are quite specific to colorectal cancer (CRC), but a method being able to detect the extraordinarily low amounts of mutants is challengeable in sensitivity. We proposed a hydrogel bead-array to digitally count CRC-specific mutants in stool at a low cost. At first, multiplex amplification of targets containing multiple mutation loci of interest is carried out by a target enriched multiplex PCR (Tem-PCR), yielding the templates qualified for emulsion PCR (emPCR). Then, after immobilizing the beads from emPCR on a glass surface, the incorporation of Cy3-dUTP into the mutant-specific probes, which are specifically hybridized with the amplified beads from emPCR, is used to color the beads coated with mutants. As all amplified beads are hybridized with the Cy5-labeled universal probe, a mutation rate is readily obtained by digitally counting the beads with different colors (yellow and red). A high specificity of the method is achieved by removing the mismatched probes in a bead-array with electrophoresis. The approach has been used to simultaneously detect 8 mutation loci within the APC, TP53, and KRAS genes in stools from eight CRC patients, and 50% of CRC patients were positively diagnosed; therefore, our method can be a potential tool for the noninvasive diagnosis of CRC.

Dye‐Free MicroRNA Quantification by Using Pyrosequencing with a Sequence‐Tagged Stem–loop RT Primer

MicroRNAs (miRNAs) are a class of endogenous, ~22-nucleotide (nt) noncoding RNAs that play an important role in the control of the developmental processes of cells by negative regulation of protein-coding gene expression. To date, there are 17 341 mature miRNAs, including 1048 human miRNAs, in the University of Manchester miRNA database (http://www. mirbase.org/). Although miRNAs represent a relatively abundant class of transcripts, their expression levels vary greatly in different tissue types and species. Analyzing miRNA expression levels in tissues or cells can supply valuable information for investigating the biological functions of miRNAs; however conventional techniques to amplify miRNAs for detection and quantification present a significant challenge because of the short length of these molecules; thus, a number of straightforward methods without the use of amplification have been developed for miRNA detection. Northern blotting 5] is the widely used standard method for analyzing miRNAs; however, relatively large amounts of starting material (RNA) are required for an assay. To improve the sensitivity of miRNA quantification, a method based on splinted ligation was developed. This exhibits approximately 50 times greater sensitivity than Northern blotting, but radioactive P labels are needed. A single-molecule method, based on the hybridization of two spectrally distinguishable LNA–DNA oligonucleotide probes (for the miRNA of interest), offers a direct miRNA assay as sensitive as 500 fm, but an expensive single-molecule detection instrument is required. For sensitive miRNA detection, amplification techniques are thus necessary. By skillfully designing detection probes, a modified “Invader” assay was developed for the quantification of miRNAs. Although 20 000 miRNAs were detected, accurate quantification of miRNAs among samples is difficult because the initial target concentration is proportional to the steady-state reaction rate of “invasive” amplification. In contrast, an miRNA assay based on real-time quantitative PCR with a stem–loop reverse transcription (RT) primer was much more quantitative, as the Ct (cycle threshold) value is inversely proportional to the amount of initial target. However, PCRs of the sample and reference targets are performed separately, and a small difference in amplification efficiency between the sample and the reference yields a large difference in the amount of final product ; this results in large inter-PCR variations. Recently, a simple and sensitive miRNA quantification method that used branched rolling-circle amplification (BRCA) was reported, but quantification based on endpoint readout seems challengeable because of the time-dependent amplification efficiency of BRCA. To achieve accurate quantification of a target miRNA in a sample, real-time monitoring of signal intensities from both a sample and a reference (quantification standard) is necessary, because the reaction rate slows down as the reaction proceeds. As the real-time detection requires a sophisticated instrument, quantification using endpoint data is preferable. In the present study, we have developed a pyrosequencing-based method for absolute quantification, and for comparing the relative miRNA expression levels in biological samples. Pyrosequencing is a well-developed technology for DNA sequencing. It uses cascade enzymatic reactions to monitor the release of inorganic pyrophosphate that results from dNTP incorporation. Because of its highly quantitative performance, pyrosequencing has been widely used for genotyping, and the analysis of DNA methylation and gene expression. Here we employed pyrosequencing technology to quantify microRNAs by quantitatively detecting sequence labels that were artificially tagged into the RT products of miRNA. Unlike mRNA, miRNA is very short and can be easily synthesized; synthesized molecules with known concentration could thus be used as a reference for quantifying miRNA in a sample. As shown in Figure 1, sequence labels for discriminating the sources of miRNA (sample or reference) are designed into the loop near to the 3’-end of the miRNA-specific RT primer, so that the 5’ end of the primer can offer a universal priming site for the following PCR. The structure of the miRNA-specific stem–loop RT primer is the same as that used by Chen’s group. After reverse transcription with the sequence-tagged RT primers, cDNA from the different sources (sample and reference) were similarly labeled with different sequences (thus, different colors in a fluorescence-based assay). To avoid PCR-bias resulting from Tm differences, the labels were designed from the same base species but with different base order. We labeled the sample-miRNA and the reference-miRNA with the sequences “catg” and “gatc” respectively ; hence, in a pyrogram (Figure 1), [a] H. Jing, Prof. Q. Song, Z. Chen, B. Zou, Prof. G. Zhou Huadong Research Institute for Medicine and Biotechnics Nanjing 210002 (China) Fax: (+ 86) 25-8451-4223 E-mail : ghzhou@nju.edu.cn [b] Prof. Q. Song, B. Zou School of Life Science and Technology, China Pharmaceutical University Nanjing 210009 (China9 [c] C. Chen, Prof. M. Zhu Model Animal Research Centre, Nanjing University Nanjing 210093 (China) [d] Z. Chen, Prof. G. Zhou Medical School, Nanjing University Nanjing 210093 (China) [e] T. Kajiyama, Prof. H. Kambara Central Research Laboratory, Hitachi, Ltd. Tokyo 185-8601 (Japan) Supporting information for this article is available on the WWW under http ://dx.doi.org/10.1002/cbic.201100023.

High-sensitivity digital radiography using an avalanche-type image pickup tube camera

A medical imaging camera with an avalanche-type image pickup tube (HARPICON) was developed for digital radiography. The camera obtains high quality images even at low doses because its sensitivity is as much as 32 times higher than that of a conventional pickup tube camera. The camera also has a wide dynamic range and a high signal-to-noise ratio because of HARPICONs gamma characteristic.

Position-Specific Gene Expression Analysis Using a Microgram Dissection Method Combined with On-Bead cDNA Library Construction

Gene expression analysis is a key technology that is used to understand living systems. Multicellular organisms, including plants, are composed of various tissues and cell types, each of which exhibits a unique gene expression pattern. However, because of their rigid cell walls, plant cells are difficult to isolate from the whole plant. Although laser dissection has been used to circumvent this problem, the plant sample needs to be fixed beforehand, which presents several problems. In the present study, we developed an alternative method to conduct highly reliable gene expression profiling. First, we assembled a dissection apparatus that used a narrow, sharpened needle to dissect out a microsample of fresh plant tissue (0.1-0.2 mm on each side) automatically from a target site within a short time frame. Then, we optimized a protocol to synthesize a high-quality cDNA library on magnetic beads using a single microsample. The cDNA library was amplified and subjected to high-throughput sequencing. In this way, a stable and reliable system was developed to conduct gene expression profiling in small regions of a plant. The system was used to analyze the gene expression patterns at successive 50 µm intervals in the shoot apex of a 4-day-old Arabidopsis seedling. Clustering analysis of the data demonstrated that two small, adjacent domains, the shoot apical meristem and the leaf primordia, were clearly distinguishable. This system should be broadly applicable in the investigation of the spatial organization of gene expression in various contexts.

Pyrosequencing-based barcodes for a dye-free multiplex bioassay

A novel dye-free labeling method for a multiplex bioassay was proposed by using short sequence-based barcodes consisting of a reporter base and repeats of two stuffer bases; then, the barcodes were quantitatively decoded by a single pyrosequencing assay without any pre-separation.

Highly Sensitive Pyrosequencing System with Polymer-Supported Enzymes for High-Throughput DNA Analysis

A highly sensitive massively parallel pyrosequencing system employing a gel matrix to immobilize enzymes at high density in microreaction chambers is demonstrated. Reducing the size of microreaction chambers in a DNA analyzer is important to achieve a high throughput utilizing a commercially available detection device or camera. A high-performance system can be attained by detecting signals from one reaction chamber with one photopixel of around several micrometers by utilizing a 1:1 image magnification. However, the use of small beads immobilizing DNA has a disadvantage in detecting luminescence because only small amounts of DNA can be immobilized on the bead surfaces for sequencing. As luminescence intensity could be enhanced by increasing the luciferase density in the chambers, we overcame this difficulty by using a gel matrix to immobilize luciferase at a high concentration in the microreaction chambers. Luminescence 1 order of magnitude higher could be observed with the new method compared to the conventional method. Consequently, the chamber size and bead size immobilizing DNA could be reduced to as small as 6.5 and 4 μm, respectively. This can be successfully applied to achieving small, inexpensive, pyrosequencing systems with high throughput.