Integrated spatial analysis of gene mutation and gene expression for understanding tumor diversity in formalin-fixed paraffin-embedded lung adenocarcinoma.

Introduction: A deeper understanding of intratumoral heterogeneity is essential for prognosis prediction or accurate treatment plan decisions in clinical practice. However, due to the cross-links and degradation of biomolecules within formalin-fixed paraffin-embedded (FFPE) specimens, it is challenging to analyze them. In this study, we aimed to optimize the simultaneous extraction of mRNA and DNA from microdissected FFPE tissues (φ = 100 µm) and apply the method to analyze tumor diversity in lung adenocarcinoma before and after erlotinib administration.

Reproducible and sensitive micro-tissue RNA sequencing from formalin-fixed paraffin-embedded tissues for spatial gene expression analysis.

Spatial transcriptome analysis of formalin-fixed paraffin-embedded (FFPE) tissues using RNA-sequencing (RNA-seq) provides interactive information on morphology and gene expression, which is useful for clinical applications. However, despite the advantages of long-term storage at room temperature, FFPE tissues may be severely damaged by methylene crosslinking and provide less gene information than fresh-frozen tissues. In this study, we proposed a sensitive FFPE micro-tissue RNA-seq method that combines the punching of tissue sections (diameter: 100 μm) and the direct construction of RNA-seq libraries.

Slow-Cycling Cancer Stem Cells Regulate Progression and Chemoresistance in Colon Cancer.

Cancer chemoresistance is often attributed to the presence of cancer stem cell (CSC)-like cells, but whether they are homogeneously chemoresistant remains unclear. We previously showed that in colon tumors, a subpopulation of CSC-like cells driven by TCF1 (TCF7), a Wnt-responsive transcription factor, were responsible for tumorigenicity. Here we demonstrate that the tumorigenic subpopulation of mouse cells exists in a slow-cycling state and identify a unique 22-gene signature that characterizes these slow-cycling CSC.

Site-specific gene expression analysis using an automated tissue micro-dissection punching system.

Site-specific gene expression analyses are important for understanding tissue functions. Despite rapid developments in DNA-related technologies, the site-specific analysis of whole genome expression for a tissue remains challenging. Thus, a new tool is required for capturing multiple tissue micro-dissections or single cells while retaining the positional information.

Prenatal Diagnosis of Chromosomal Aneuploidies by Quantitative Pyrosequencing®.

One of the major reasons for pregnant women to ask for prenatal diagnosis is to detect fetal chromosomal aneuploidies. Analysis of allele ratios of SNPs has been used for prenatal detection of fetal aneuploidies using MALDI-TOF mass spectrometry (MS). However, quantitative SNP genotyping by MALDI-TOF MS is challenging.

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.

Involvement of Ca2+ in Vacuole Degradation Caused by a Rapid Temperature Decrease in Saintpaulia Palisade Cells: A Case of Gene Expression Analysis in a Specialized Small Tissue.

Saintpaulia (African violet) leaves are known to be damaged by a rapid temperature decrease when cold water is applied to the leaf surface; the injury is ascribed to the chloroplast damage caused by the cytosolic pH decrease following the degradation of the vacuolar membrane in the palisade cells. In this report, we present evidence for the involvement of Ca(2+) in facilitating the collapse of the vacuolar membrane and in turn in the temperature sensitivity of Saintpaulia leaves. In the presence of a Ca(2+) chelator (EGTA) or certain Ca(2+) channel inhibitors (Gd(3+) or La(3+)) but not others (verapamil or nifedipine), the pH of the vacuole, monitored through BCECF (2',7'-bis(carboxyethyl)-4 or 5-carboxyfluorescein) fluorescence, did not increase in response to a rapid temperature drop.

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.

Self-oriented immobilization of DNA polymerase tagged by titanium-binding peptide motif.

We developed a titanium-binding-peptide-1 (TBP-1)-tagged DNA polymerase, for self-oriented immobilization onto a titanium oxide (TiO2) substrate. The enzymatic function of a polymerase immobilized on a solid state device is strongly dependent on the orientation of the enzyme. The TBP-tagged DNA polymerase, which was derived from a hyperthermophilic archaeon, was designed to incorporate the RKLPDA peptide at the N-terminus, and synthesized by translation processes in Escherichia coli (E.

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 highly sensitive and accurate gene expression analysis by sequencing ("bead-seq") for a single cell.

Analyses of gene expressions in single cells are important for understanding detailed biological phenomena. Here, a highly sensitive and accurate method by sequencing (called "bead-seq") to obtain a whole gene expression profile for a single cell is proposed. A key feature of the method is to use a complementary DNA (cDNA) library on magnetic beads, which enables adding washing steps to remove residual reagents in a sample preparation process.

Non-biased and efficient global amplification of a single-cell cDNA library.

Analysis of single-cell gene expression promises a more precise understanding of molecular mechanisms of a living system. Most techniques only allow studies of the expressions for limited numbers of gene species. When amplification of cDNA was carried out for analysing more genes, amplification biases were frequently reported.

Improvement of LATE-PCR to allow single-cell analysis by pyrosequencing.

Nucleic acid analysis in a single cell is very important, but the extremely small amount of template in a single cell requires a detection method more sensitive than the conventional method. In this paper, we describe a novel assay allowing a single cell genotyping by coupling improved linear-after-the-exponential-PCR (imLATE-PCR) on a modified glass slide with highly sensitive pyrosequencing. Due to the significantly increased yield of ssDNA in imLATE-PCR amplicons, it is possible to employ pyrosequencing to sequence the products from 1 μL chip PCR which directly used a single cell as the starting material.

Hedgehog-Gli activators direct osteo-chondrogenic function of bone morphogenetic protein toward osteogenesis in the perichondrium.

Specification of progenitors into the osteoblast lineage is an essential event for skeletogenesis. During endochondral ossification, cells in the perichondrium give rise to osteoblast precursors. Hedgehog (Hh) and bone morphogenetic protein (BMP) are suggested to regulate the commitment of these cells.

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).

Multiplex loop-mediated isothermal amplification detection by sequence-based barcodes coupled with nicking endonuclease-mediated pyrosequencing.

The loop-mediated isothermal amplification (LAMP) is a well-developed method for replicating a targeted DNA sequence with a high specificity, but multiplex LAMP detection is difficult because LAMP amplicons are very complicated in structure. To allow simultaneous detection of multiple LAMP products, a series of target-specific barcodes were designed and tagged in LAMP amplicons by FIP primers. The targeted barcodes were decoded by pyrosequencing on nicked LAMP amplicons.

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.

Optimization of pyrosequencing reads by superior successive incorporation efficiency of improved 2'-deoxyadenosine-5'-triphosphate analogs.

Conventional pyrosequencing using 2'-deoxyadenosine-5'-O-(1-thiotriphosphate) (dATPαS) is problematic due to the high cost of the substrate (dATPαS) and deterioration in the accuracy of incorporation to read through poly(T) regions. One reason for these problems is that dATPαS has a sulfur on the α-phosphate and also has isomers (Sp and Rp). To solve these problems, 11 nucleotide substrates, which could replace dATPαS in pyrosequencing, were newly synthesized.

Digital analysis of the expression levels of multiple colorectal cancer-related genes by multiplexed digital-PCR coupled with hydrogel bead-array.

To digitally analyze expression levels of multiple genes in one reaction, we proposed a method termed as 'MDHB' (Multiplexed Digital-PCR coupled with Hydrogel Bead-array). The template for bead-based emulsion PCR (emPCR) was prepared by reverse transcription using sequence-tagged primers. The beads recovered from emPCR were immobilized with hydrogel to form a single-bead layer on a chip, and then decoded by gene-specific probe hybridization and Cy3-dUTP based primer extension reaction.

Highly sensitive pyrosequencing based on the capture of free adenosine 5' phosphosulfate with adenosine triphosphate sulfurylase.

In pyrosequencing chemistry, four cascade enzymatic reactions with the catalysis of polymerase, adenosine triphosphate (ATP) sulfurylase, luciferase, and apyrase are employed. The sensitivity of pyrosequencing mainly depends on the concentration of luciferase which catalyzes a photoemission reaction. However, the side-reaction of adenosine 5' phosphosulfate (APS, an analogue of ATP) with luciferase resulted in an unavoidable background signal; hence, the sensitivity cannot be much higher due to the simultaneous increase of the background signal when a larger amount of luciferase is used.

Gene expression analysis on a photodiode array-based bioluminescence analyzer by using sensitivity-improved SRPP.

Most methods used for gene expression analysis are based on dye-labeling, which requires costly instruments. Recently a dye-free gene expression analysis method-SRPP (Sequence-tagged reverse-transcription polymerase chain reaction coupled with pyrosequencing) was developed to compare relative gene expression levels in different tissues, but the throughput of the SRPP assay is very limited due to the use of a photomultiplier tube (PMT)-based pyrosequencer for the detection. To increase the throughput of the SRPP assay, an inexpensive photodiode (PD) array-based bioluminescence analyzer (termed as "PD-based pyrosequencer") was coupled to SRPP; however the low sensitivity of PD limited the wide application of SRPP.

Pyrosequencing on nicked dsDNA generated by nicking endonucleases.

Although the pyrosequencing method is simple and fast, the step of ssDNA preparation increases the cost, labor, and cross-contamination risk. In this paper, we proposed a method enabling pyrosequencing directly on dsDNA digested by nicking endonucleases (NEases). Recognition sequence of NEases was introduced using artificially mismatched bases in a PCR primer (in the case of genotyping) or a reverse-transcription primer (in the case of gene expression analysis).

Highly sensitive mutation detection based on digital amplification coupled with hydrogel bead-array.

In order to detect small amounts of mutants for early cancer diagnosis, we have developed the novel method of using amplicon-coated microbeads and single-molecule-PCR in water-in-oil emulsions, which we coupled with a new detection platform, the hydrogel bead-array, a 3-D polyacrylamide gel network used as a carrier to immobilize the beads.