Rongqin Ke

National Huaqiao University

RNA in situ hybridization reveals the abundance and location of gene expression in cells or tissues, providing a technical basis for the clinical diagnosis of diseases. In this chapter, we show a “V” shape probe-mediated single-molecule chromogenic in situ hybridization (vsmCISH) technique for bright-field visualization of individual RNA molecules....

Multiplexed in situ RNA imaging offers new opportunities for gene expression profiling by providing high-throughput spatial information. In this work, we present a cyclic combinatorial fluorescent in situ hybridization (combFISH) assay to achieve multiplexed detection of RNA in cell cultures and tissues. Specifically, multiplexing is achieved throu...

Endogenous retroviruses (ERVs) are ancient retroviral remnants integrated in host genomes, and commonly deleted through unequal homologous recombination, leaving solitary long terminal repeats (solo-LTRs). This study, analysing the genomes of 362 bird species and their reptilian and mammalian outgroups, reveals an unusually higher level of solo-LTR...

Spatial transcriptomics (ST) is featured by high‐throughput gene expression profiling within their native cell and tissue context, offering a means to investigate gene regulatory networks in tissue microenvironment. In situ sequencing (ISS) is an imaging‐based ST technology that simultaneously detects hundreds to thousands of genes at subcellular r...

Loss of refreshment in nucleus pulposus (NP) cellularity leads to intervertebral disc (IVD) degeneration. Nevertheless, the cellular sequence of NP cell differentiation remains unclear, although an increasing body of literature has identified markers of NP progenitor cells (NPPCs). Notably, due to their fragility, the physical enrichment of NP‐deri...

The heart is an autoimmune-prone organ. It is crucial for the heart to keep injury-induced autoimmunity in check to avoid autoimmune-mediated inflammatory disease. However, little is known about how injury-induced autoimmunity is constrained in hearts. Here, we reveal an unknown intramyocardial immunosuppressive program driven by Tbx1, a DiGeorge s...

Visualizing spatial patterns of gene expression by optical microscopy at single-molecule resolution represents a long-standing challenge for imaging and molecular engineering technologies. In this study, we developed a method for visualizing mRNA with duplex capability by optical microscopy using rolling circle amplification with streptavidin-modif...

Although RNA plays a vital role in the process of gene expression, it is less used as an in situ biomarker for clinical diagnostics compared to DNA and protein. This is mainly due to technical challenges caused by the low expression level and easy degradation of RNA molecules themselves. To tackle this issue, methods that are sensitive and specific...

RNA plays a vital role in the physiological and pathological processes of cells and tissues. However, RNA in situ hybridization applications in clinical diagnostics are still limited to a few examples. In this study, we developed a novel in situ hybridization assay for human papillomavirus (HPV) E6/E7 mRNA by taking advantage of specific padlock pr...

Spatial transcriptomics enables the study of localization-indexed gene expression activity in tissues, providing the transcriptional landscape that in turn indicates the potential regulatory networks of gene expression. In situ sequencing (ISS) is a targeted spatial transcriptomic technique, based on padlock probe and rolling circle amplification c...

Synthetic single‐stranded (ss) DNA is a cornerstone for life and materials science, yet the purity, quantity, length, and customizability of synthetic DNA are still limiting in various applications. Here, we present PECAN, paired‐end cutting assisted by DNAzymes (DNA enzymes or deoxyribozymes), which enables mass production of ssDNA of arbitrary se...

Synthetic single‐stranded (ss) DNA is a cornerstone for life and materials science, yet the purity, quantity, length, and customizability of synthetic DNA are still limiting in various applications. Here we present PECAN, paired‐end cutting assisted by DNAzymes (DNA enzymes or deoxyribozymes), which enables mass production of ssDNA of arbitrary seq...

Spatial transcriptomics enables the study of localization-indexed gene expression activity in tissues, providing the transcriptional landscape that in turn indicates the potential regulatory networks of gene expression. In situ sequencing (ISS) is a targeted spatial transcriptomic technique that is based on padlock probe and rolling circle amplific...

The molecular heterogeneity of cancer is one of the major causes of drug resistance that leads to treatment failure. Thus, better understanding the heterogeneity of cancer will contribute to more precise diagnosis and improved patient outcomes. Although single-cell sequencing has become an important tool for investigating tumor heterogeneity recent...

Preparation of sufficient mouse Leydig cells (LCs) with high purity is a prerequisite for investigations of the biological/pathological functions of LCs in mouse models. Density gradient centrifugation based on discontinuous Percoll gradients is an effective method (defined as regular method) for LC isolation. In this study, we developed two modifi...

Precise generation of excitatory neurons and inhibitory interneurons is crucial for proper formation and function of neural circuits in the mammalian brain. Because of the size and complexity of the human brain, it is a challenge to reveal the rich diversity of interneurons. To decipher origin and diversity of interneurons in the human fetal subpal...

Glucose consumption is generally increased in tumor cells to support tumor growth. Interestingly, we report that glycogen accumulation is a key initiating oncogenic event during liver malignant transformation. We found that glucose-6-phosphatase (G6PC) catalyzing the last step of glycogenolysis is frequently downregulated to augment glucose storage...

Liquid biopsy with circulating tumor DNA (ctDNA) profiling by next-generation sequencing holds great promise to revolutionize clinical oncology. It relies on the basis that ctDNA represents the real-time status of the tumor genome which contains information of genetic alterations. Compared to tissue biopsy, liquid biopsy possesses great advantages...

As a highly prevalent disease among women worldwide, breast cancer remains in urgent need of further elucidation its molecular mechanisms to improve the patient outcomes. Identifying hub genes involved in the pathogenesis and progression of breast cancer can potentially help to unveil mechanism and also provide novel diagnostic and prognostic marke...

As a highly prevalent disease among women worldwide, breast cancer remains in urgent need of further elucidation its molecular mechanisms to improve the patient outcomes. Identifying hub genes involved in the pathogenesis and progression of breast cancer can potentially help to unveil mechanism and also provide novel diagnostic and prognostic marke...

Klinefelter syndrome (KS) is one of the most frequent genetic abnormalities and the leading genetic cause of non-obstructive azoospermia. The breeding of mouse models of KS and their study are essential to advance our knowledge of the pathologic mechanism. Karyotyping and fluorescence in situ hybridization are reliable methods for identifying chrom...

An amplification-based single-molecule fluorescence in situ hybridization (asmFISH) assay is introduced that exploits improved probe design for highly specific imaging of individual transcripts in fixed cells and tissues. In this method, a pair of DNA ligation probes are ligated on RNA templates upon specific hybridization, followed by probe circul...

Background As a highly prevalent tumor disease worldwide, Further elucidation of the molecular mechanisms of the occurrence, development and prognosis of breast cancer remain an urgent need. Identifying hub genes involved in these pathogenesis and progression can potentially help to unveil its mechanism and provide novel diagnostic and prognostic m...

Conventional flow cytometry has been widely used for high throughput single cell gene expression analysis using specific antibody staining. However, this is limited by the availability of high-quality antibodies. We developed a novel flow cytometry RNA detection technique termed RCA-Flow for single cell RNA expression analysis. We showed it is able...

The emerging in situ RNA sequencing technologies which can capture and amplify RNA within the original tissues provides efficient solution for producing spatial expression map from dozens to thousands of genes. Most of in situ RNA-seq strategies developed recently infer the expression patterns based on the fluorescence signals from the images taken...

MicroRNAs (miRNAs) are key regulators of gene expression at the posttranscriptional level. Precisely profiling of miRNA expression will help us to better understand their roles in normal and diseased cells and tissues. Here we describe in situ miRNA detection by padlock probing and miRNA target-primed rolling circle amplification. We optimized our...

Visualization of RNA molecules in situ helps to better understand the functions of expressed genes. Currently, most conventional in situ hybridization methods for visualization of individual RNAs are based on fluorescence detection. Herein we present a chromogenic in situ hybridization protocol for visualization of single RNA molecules in fixed cel...

Ubiquitin-specific protease (USP) 6 is a hominoid deubiquitinating enzyme previously implicated in intellectual disability and autism spectrum disorder. Although these findings link USP6 to higher brain function, potential roles for USP6 in cognition have not been investigated. Here, we report that USP6 is highly expressed in induced human neurons...

Visualization of gene expression at single RNA molecular level represents a great challenge to both imaging technologies and molecular engineering. Here we show a single molecule chromogenic in situ hybridization (smCISH) assay that enables counting and localizing individual RNA molecules in fixed cells and tissue under bright-field microscopy. Our...

In this review, we discuss the emergence of 4th generation sequencing technologies that preserve the spatial coordinates of RNA and DNA sequences with up to subcellular resolution thus enabling back mapping of sequencing reads to the original histological context. This information is used for example in two current large-scale projects that aim to...

The REvolutionary Approaches and Devices for Nucleic Acid analysis (READNA) project received funding from the European Commission for 41/2 years. The objectives of the project revolved around technological developments in nucleic acid analysis. The project partners have discovered, created and developed a huge body of insights into nucleic acid ana...

We present a method for in situ detection of individual mRNA molecules in preserved tissue and cells. The method not only enables digital quantification of mRNA molecules but also provides the localization information of the detected mRNA molecules at subcellular resolution. With this technology, it is possible to study heterogeneity of gene expres...

Profiling of gene expression is necessary to study the function of cells, organs and ultimately organisms, in health and disease. However, the currently available mRNA sequencing methods are limited to homogenized tissue samples. Therefore, the obtained information represents either the average expression profile of the full tissue sample or expres...

A europium nanoparticle-based lateral flow immunoassay for highly sensitive detection of chloramphenicol residue was developed. The detection result could be either qualitatively resolved with naked eye or quantitatively analyzed with the assistance of a digital camera. In the qualitative mode, the limit of detection (LOD) was found to be 0.25 ng/m...

Proximity ligation assay (PLA) has been proven to be a robust protein detection method. The technique is characterized by high sensitivity and specificity, but the assay precision is probably limited by the PCR readout. To investigate this potential limitation and to improve precision, we developed a digital proximity ligation assay for protein mea...

Tissue gene expression profiling is performed on homogenates or on populations of isolated single cells to resolve molecular states of different cell types. In both approaches, histological context is lost. We have developed an in situ sequencing method for parallel targeted analysis of short RNA fragments in morphologically preserved cells and tis...

Genome rearrangements have important effects on bacterial phenotypes and influence the evolution of bacterial genomes. Conventional strategies for characterizing rearrangements in bacterial genomes rely on comparisons of sequenced genomes from related species. However, the spectra of spontaneous rearrangements in supposedly homogenous and clonal ba...

Principle of rolling circle amplification (RCA). 1a) Padlock probes and connector oligonucleotides were added to samples and hybridized to the correct template. 1b) Padlock probes and connector oligonucleotides were then ligated by DNA ligase to form a completed DNA circle. 2) Ligated padlock probes were amplified by RCA. 3a) At the presence of res...

Illustration of split mapping and classification for putative rearrangements. The split read has the prefix and suffix mapped to different locations on the reference genome. The prefix and suffix are defined as the first and second split segments coming in the read and have no indication of the mapping orientations. The basic signatures include (i)...

A magnetic-nanobead-based, substrate-free method for the sensitive detection of spores in an immunoassay format is presented. The method is shown to detect Bacillus globigii spores, the non-pathogenic simulant of Bacillus anthracis, with a limit-of-detection of 50 spores with a reaction time of 135 min. The study shows the versatility of magnetic n...

Detection and identification of pathogens in environmental samples for biosecurity applications are challenging due to the strict requirements on specificity, sensitivity and time. We have developed a concept for quick, specific and sensitive pathogen identification in environmental samples. Target identification is realized by padlock- and proximi...

Schematic description of the optical set-up of the detection instrument. A) Optical pathway fluorescence excitation schematics. a) 640 nm laser, b) 532 nm laser, c) 491 nm laser, d) beam expenders, e) laser mirrors, f) 532/640 nm laser beam combiner, g) 491/532/640 nm laser beam combiner, h) beam shaping optics, i) triple-laser pass dichroic mirror...

Observed digestion and inactivation properties of the tested restriction enzymes. (DOCX)

A part of a scanned image showing the RCPs. Each RCP is visualized as a bright dot, and is therefore counted as ‘1’. The figure is not drawn to scale. (TIF)

Lambda DNA Digestion products with and without preceding incubation at 65°C. For both lanes 2 in A and both lanes 10 in B: 1 kb ladder. Both lanes 3 in A: undigested Lambda DNA. Under each enzyme legend in A and B, the first lane contains DNA digested for 5 min after incubation at 65°C 5 min and the second lane contains DNA digested for 5 min after...

Lambda DNA Digestion products with and without preceding incubation at 65°C using different incubation periods. First and last lane in both rows: 1 kb ladder. Lane 2, both rows: undigested Lambda DNA. Under each enzyme legend, the first three lanes contain DNA digested for 5 min after incubation at 65°C for 1, 2 and 4 min respectively. The followin...

Detection of E coli genome by quantitative PCR. A) A dilution series of genomic DNA isolated from E coli was analyzed by quantitative PCR. B) Prepared samples from spreading by the ASAP air sampler detected by quantitative PCR. The negative control sample is water. The standard deviations are from triplicate samples. (TIF)

Supporting materials and methods. (DOCX)

A picture showing how the dissemination equipment and the air sampling system work. (TIF)

We developed a molecular diagnostic method for detection of RNA virus based on padlock probes and colorimetric readout. The feasibility of our approach was demonstrated by using detection of Crimean-Congo hemorrhagic fever (CCHF) virus as a model. Compared with conventional PCR-based methods, our approach does not require advanced equipment, involv...

We present a new type of enzyme-antibody conjugate that simplifies the labeling procedure and increases the sensitivity of enzyme-linked immunosorbent assay (ELISA). The conjugates were prepared through layer-by-layer immobilization of enzyme and antibody on a silica nanoparticle scaffold. A maximal amount of enzyme was immobilized on the nanoparti...