Subcellular Localization of MicroRNAs by MicroRNA In Situ Hybridization (miR-ISH).

MicroRNAs (miRNAs) are 22-nucleotide RNA sequences that regulate up to 60% of the mammalian transcriptome. Although canonical miRNA-induced silencing complex-mediated messenger RNA degradation occurs in the cytoplasm, miRNAs have been described in other subcellular compartments with potentially novel functions. Currently, there are limited methodologies for visualizing RNA locations within cells to elucidate mechanisms and pathways of miRNA biogenesis, transport, and function. Here, we describe a simple and rapid miRNA in situ hybridization method that can be combined with standard immunofluorescence procedures for subcellular localization of mature and precursor miRNAs.

Practical application of non-contact alternating current electric field mixing for reagent-saving in situ hybridisation of HER2.

AIMS: Human epidermal growth factor receptor 2 (HER2)-targeted agents are effective against HER2-positive breast cancers. However, their lack of survival benefit in HER2-negative patients as well as their toxic effects and high cost highlight the need for accurate assessment of HER2 status. Our aim was to evaluate the clinical utility of a reagent-saving in situ hybridisation (Saving ISH) that facilitates hybridisation and saves HER2/chromosome enumeration probe by taking advantage of the non-contact mixing effect of an alternating current (AC) electric field. METHODS: With a new device, we apply a high-voltage, low-frequency AC electric field to the tissue sections, which mixes the probe within microdroplets as the voltage is switched on and off. Specimens (n=113) from patients with breast cancers identified immunohistochemically as HER2 0/1(+), (2+) or (3+) were used. The specimens were all tested using conventional dual ISH (DISH), DISH with an automated slide stainer (ASS) and Saving ISH (1:1-1:3 dilution). RESULTS: The Saving ISH with 1:2 probe dilution produced stable results with less non-specific staining while using smaller amounts of probe. The accuracy of HER2 status with Saving ISH was equal to standard. We found 96.4% agreement between DISH using ASS and Saving ISH (kappa coefficient=0.912). CONCLUSIONS: These results suggest reagent-saving HER2 ISH could be used as a clinical tool for accurate and stable HER2 assessment, even when reagent concentrations vary.

Analysis of mRNA, miRNA, and DNA in Bone Cells by RT-qPCR and In Situ Hybridization.

The aim of this chapter is to describe a method used to evaluate gene expression and microRNAs (miRNAs) in bone cells or tissue using Reverse transcription and quantitative Polymerase Chain Reaction (RT-qPCR), and a method to assess chromogenic in situ hybridization (CISH) on Formalin Fixed Paraffin Embedded (FFPE ) mouse bone tissue to detect both DNA and mRNA transcripts using the double digoxigenin (DIG) locked nucleic acid (LNA™) probes .

Predictive Marker: HER2 in Esophageal Adenocarcinoma.

HER2 positivity is based on the fundamental principle of amplification of the human epidermal growth factor receptor 2 (HER2) gene resulting in overexpression of the protein products . Arising from that a "HER2-positive cancer" is one that shows HER2 gene amplification and resultant protein expression as demonstrated by in situ hybridization and immunohistochemistry, respectively. Testing of the HER2 status is crucial to ensure selection of the correct patient who may benefit from target therapy for esophageal adenocarcinoma. Accurate testing is dependent on several pre-analytical and analytical factors including sample selection, laboratory techniques, and accurate interpretation of HER2 test results.

Detection and Typing of "Candidatus Phytoplasma " spp. in Host DNA Extracts Using Oligonucleotide-Coupled Fluorescent Microspheres.

The use of oligonucleotide-coupled fluorescent microspheres is a rapid, sequencing-independent, and reliable way to diagnose bacterial diseases. Previously described applications of oligonucleotide-coupled fluorescent microspheres for the detection and identification of bacteria in human clinical samples have been successfully adapted to detect and differentiate "Ca. Phytoplasma" species using as a target the chaperonin 60-encoding gene. In this chapter, we describe in detail the design and validation of oligonucleotide capture probes, and their application in the assay aiming to differentiate phytoplasma strains infecting Brassica napus and Camelina sativa plants grown in the same geographic location at the same time.

Paper analytical devices for dynamic evaluation of cell surface N-glycan expression via a bimodal biosensor based on multibranched hybridization chain reaction amplification.

A novel colorimetric/fluorescence bimodal lab-on-paper cyto-device was fabricated based on concanavalin A (Con A)-integrating multibranched hybridization chain reaction (mHCR). The product of mHCR was modified PtCu nanochains (colorimetric signal label) and graphene quantum dot (fluorescence signal label) for in situ and dynamically evaluating cell surface N-glycan expression. In this strategy, preliminary detection was carried out through colorimetric method, if needed, then the fluorescence method was applied for a precise determination. Au-Ag-paper devices increased the surface areas and active sites for immobilizing larger amount of aptamers, and then specifically and efficiently captured more cancer cells. Moreover, it could effectively reduce the paper background fluorescence. Due to the specific recognition of Con A with mannose and the effective signal amplification of mHCR, the proposed strategy exhibited excellent high sensitivity for the cytosensing of MCF-7 cells ranging from 100 to 1.0×10(7) and 80-5.0×10(7) cellsmL(-1) with the detection limit of 33 and 26 cellsmL(-1) for colorimetric and fluorescence, respectively. More importantly, this strategy was successfully applied to dynamically monitor cell-surface multi-glycans expression on living cells under external stimuli of inhibitors as well as for N-glycan expression inhibitor screening. These results implied that this biosensor has potential in studying complex native glycan-related biological processes and elucidating the N-glycan-related diseases in biological and physiological processes.

Fiber optofluidic biosensor for the label-free detection of DNA hybridization and methylation based on an in-line tunable mode coupler.

An optical fiber optofluidic biosensor for the detection of DNA hybridization and methylation has been proposed and experimentally demonstrated. An in-line fiber Michelson interferometer was formed in the photonic crystal fiber. A micrhole in the collapsed region, which combined the tunable mode coupler and optofluidic channel, was fabricated by using femtosecond laser micromachining. The mode field diameter of the guided light is changed with the refractive index in the optofluidic channel, which results in the tunable coupling ratio. Label-free detections of the DNA hybridization and methylation have been experimentally demonstrated. The probe single stranded DNA (ssDNA) was bound with the surface of the optofluidic channel through the Poly-l-lysine layer, and the hybridization between a short 22-mer probe ssDNA and a complementary target ssDNA was carried out and detected by interrogating the fringe visibility of the reflection spectrum. Then, the DNA methylation was also detected through the binding between the methylated DNA and the 5-methylcytosine (5-mC) monoclonal antibody. The experiments results demonstrate that the limit of detection of 5nM is achieved, establishing the tunable mode coupler as a sensitive and versatile biosensor. The sensitive optical fiber optofluidic biosensor possesses high specificity and low temperature cross-sensitivity.

A sensitive impedimetric platform biosensing protein: Insoluble precipitates based on the biocatalysis of manganese(III) meso-tetrakis (4-N-methylpyridiniumyl)-porphyrinin in HCR-assisted dsDNA.

In this study, a sensitive biosensing interface for protein was reported based on nonconductive insoluble precipitates (IPs) by the biocatalysis of manganese(III) meso-tetrakis (4-N-methylpyridiniumyl)-porphyrin (MnTMPyP), which was intercalated into formed double-strand DNA (dsDNA) scaffold triggered by hybridization chain reaction (HCR). In the proposed impedimetric aptasensor, carcinoembryonic antigen (CEA) and its aptamer were used as testing model. PtPd nanowires (PtPdNWs) with large surface area and superior conductivity were employed as nanocarriers to greatly immobilize biomolecules (e.g. CEA aptamers). Then, two DNA hairpins H1 and H2 were introduced to trigger HCR with the assistance of DNA initiator, resulting in the formation of a long dsDNA scaffold. Meanwhile, mimicking enzyme MnTMPyP molecules were embedded into the resultant dsDNA, in situ generating the complex MnTMPyP-dsDNA with peroxidase-like activity. Under the biocatalysis of MnTMPyP-dsDNA, 3,3-diaminobenzidine (DAB) was oxidized to form nonconductive IPs. As a result, the electron transfer between electrode interface and redox probe was vastly hindered, leading to the significant amplification of electrochemical impedimetric signal. So, greatly improved analytical performances of the proposed aptasensor were achieved with a detection limit as low as 0.030pgmL(-1). And the successful assay of CEA in human serum samples enabled the developed biosensing platform to have promising potential in bioanalysis.

Label-free in-situ real-time DNA hybridization kinetics detection employing microfiber-assisted Mach-Zehnder interferometer.

A label-free DNA biosensor based on microfiber-assisted Mach-Zehnder interferometer (MAMZI) for in-situ real-time DNA hybridization kinetics detection has been proposed and experimentally demonstrated. A microfiber of hundreds of microns in length is fabricated by tapering a segment of standard single-mode fiber (SMF) to construct the U-shaped microcavity between the lead-in and lead-out SMFs. Thanks to the mode field mismatching between the SMF and microfiber, the incident guided mode light would separate into two beams that respectively propagate in the air microcavity and the microfiber. Consequently, interference between different light modes would occur at the joint between the microfiber and the lead-out SMF. Experimental results indicate that owing to the participation of opening cavity modes in the modal interference process, the interferometric spectrum of our proposed microcavity sensor is highly sensitive to the variation of environmental refractive index (RI), especially for the RI range around 1.34 which is useful for most biological applications. The microfiber functionalization is achieved by stepwise modifying the microfiber with monolayer Poly-l-lysine (PLL) and single-stranded DNA (ssDNA) probes to produce the sensitive surface that could uniquely attach specific target ssDNAs. The fiber surface functionalization as well as DNA hybridization processes have been experimentally investigated for different target ssDNA solutions in real time. The interferometric transmission spectrum shows large wavelength shift for different biological phases, and a detection limit conservatively down to 0.0001pmol/µL has been acquired by employing the U-shaped microcavity of 176.88µm in length. Our proposed DNA biosensor possesses several advantages such as compact size, ease of fabrication, and strong response for DNA hybridization, which make it a promising candidate for potential applications in such rapidly expanding areas as medical diagnosis, cancer screenings, medicine examination and environmental engineering, etc.

Biosensor for label-free DNA quantification based on functionalized LPGs.

A label-free fiber optic biosensor based on a long period grating (LPG) and a basic optical interrogation scheme using off the shelf components is used for the detection of in-situ DNA hybridization. A new methodology is proposed for the determination of the spectral position of the LPG mode resonance. The experimental limit of detection obtained for the DNA was 62±2nM and the limit of quantification was 209±7nM. The sample specificity was experimentally demonstrated using DNA targets with different base mismatches relatively to the probe and was found that the system has a single base mismatch selectivity.

Electrochemical sensor for glutathione detection based on mercury ion triggered hybridization chain reaction signal amplification.

In this work, we design a new simple and highly sensitive strategy for electrochemical detection of glutathione (GSH) via mercury ion (Hg(2+)) triggered hybridization chain reaction (HCR) signal amplification. It is observed that in the absence of GSH, a specific thymine-Hg(2+)-thymine (T-Hg(2+)-T) coordination can fold into hairpin structures. While in the presence of GSH, it thus can be chelated with Hg(2+), resulting in Hg(2+) released from the T-Hg(2+)-T hairpin complex which then forms into ssDNA structure to further hybridize with the surface-immobilized capture DNA probe on the gold electrode with a sticky tail left. The presence of two hairpin helper probes through HCR leads to the formation of extended dsDNA superstructure on the electrode surface, which therefore causes the intercalation of numerous electroactive species ([Ru(NH3)6](3+)) into the dsDNA grooves, followed by a significantly amplified signal output whose intensity is related to the concentration of the GSH. Taking advantage of merits of enzyme-free amplification power of the HCR, the inherent high sensitivity of the electrochemical technique, and label-free detection which utilizes an electroactive species as a signaling molecule that binds to the anionic phosphate backbone of DNA strands via electrostatic force, not only does the proposed strategy enable sensitive detection of GSH, but show high selectivity against other amino acid, making our method a simple and sensitive addition to the amplified GSH detection.

The origin of in situ hybridization - A personal history.

In situ hybridization is the technique by which specific RNA or DNA molecules are detected in cytological preparations. Basically it involves formation of a hybrid molecule between an endogenous single-stranded RNA or DNA in the cell and a complementary single-stranded RNA or DNA probe. In its original form the probe was labeled with (3)H and the hybrid was detected by autoradiography. The first successful experiments in 1968 involved detection of the highly amplified ribosomal DNA in oocytes of the frog Xenopus, followed soon after by the reiterated "satellite DNA" in mouse and Drosophila chromosomes. Fluorescent probes were developed about ten years later.

Coupling hybridization chain reaction with catalytic hairpin assembly enables non-enzymatic and sensitive fluorescent detection of microRNA cancer biomarkers.

The identification and quantification of sequence-specific microRNAs (miRNAs) plays an important role in early diagnosis of different diseases. In this work, by integrating two independent signal amplification approaches, hybridization chain reaction and catalytic hairpin assembly, we report an enzyme-free and dual amplified approach for highly sensitive detection of a human prostate cancer biomarker, miR-141. The presence of miR-141 triggers the self-assembly of two hairpin DNAs into dsDNA polymers, which co-localize two split segments of ssDNA into proximity. Subsequently, these co-localized ssDNA sequences further act as triggers to initiate catalytic assembly of two fluorescently quenched hairpin DNAs to form numerous dsDNA strands, resulting in the recovery of the fluorescent emissions and remarkably amplified signals for highly sensitive detection of miR-141 down to 0.3 fM. In addition, this method is also selective for the target miRNA against other control sequences. With the advantages of high sensitivity and nanomaterial/enzyme-free detection format, the developed method can be a general sensing platform for the detection of trace amounts of sequence-specific nucleic acid targets.

ß-Cyclodextrin functionalized graphene as a highly conductive and multi-site platform for DNA immobilization and ultrasensitive sensing detection.

A versatile nanocomposite containing ß-cyclodextrin and graphene (CD-GR) was prepared through a simple chemical reduction method. The characterization experiments show that the nanocomposite remains the flake-like morphology of GR, but its solubility and stability in aqueous solution are greatly improved. Then the nanocomposite was modified at glassy carbon electrode (GCE) surface, and was used as a functional matrix for the covalent immobilization of probe DNA using 2,4,6-trichloro-1,3,5-triazine (TCT) as the crosslinker. Due to the synergetic effect of large surface area of GR and rich hydroxyl of CD, the probe density for the developed biosensor was determined to as high as 3.82×10(13) molecules cm(-2). Meanwhile, the biosensor shows high hybridization efficiency and hybridization kinetic. When the biosensor was applied for the impedance-based hybridization test, a wide linear range from 1.0×10(-16) to 1.0×10(-12) M and an ultralow detection limit of 3.4×10(-17) M were achieved. The biosensor also displays excellent stability, selectivity, and reproducibility.

Enhanced electrochemical recognition of double-stranded DNA by using hybridization chain reaction and positively charged gold nanoparticles.

Enhanced sequence-specific recognition of double-stranded DNA (dsDNA) was realized by using hybridization chain reaction (HCR) and positively charged gold nanoparticles ((+)AuNPs) dual signal amplification. To construct such a sensor, capture probe was initially assembled onto gold electrode surface. Upon addition of dsDNA, sandwiched DNA complex was formed between the capture probe and the detection probe, then another exposed part of the detection probe opened two alternating DNA hairpins (H1 and H2) in turn and initiated HCR to form a double-helix. Meantime, (+)AuNPs were electrostatically adsorbed onto such double-helix to amplify the electrochemical signal. Upon optimal conditions, the electronic signals of ferrocene (Fc) that modified on H1 and H2 increased linearly with increasing dsDNA concentration over the range from 15 pM to 1.0 nM, with a detection limit of 2.6 pM. Moreover, the proposed method showed good sequence specificity for dsDNA recognition.

An exonuclease-assisted amplification electrochemical aptasensor for Hg(2+) detection based on hybridization chain reaction.

In this work, a novel electrochemical aptasensor was developed for Hg(2+) detection based on exonuclease-assisted target recycling and hybridization chain reaction (HCR) dual signal amplification strategy. The presence of Hg(2+) induced the T-rich DNA partly folded into duplex-like structure via the Hg(2+) mediated T-Hg(2+)-T base pairs, which triggered the activity of exonuclease III (Exo III). Exo III selectively digested the double-strand DNA containing multiple T-Hg(2+)-T base pairs from its 3'-end, the released Hg(2+) participated analyte recycle. With each digestion cycle, a digestion product named as help DNA was obtained, which acted as a linkage between the capture DNA and auxiliary DNA. The presence of help DNA and two auxiliary DNA collectively facilitated successful HCR process and formed long double-stranded DNA. [Ru(NH3)6](3+) was used as redox indicator, which electrostatically bound to the double strands and produced an electrochemical signal. Exo III-assisted target recycling and HCR dual amplification significantly improved the sensitivity for Hg(2+) with a detection limit of 0.12 pM (S/N=3). Furthermore, the proposed aptasensor had a promising potential for the application of Hg(2+) detection in real aquatic sample analysis.

Target-regulated proximity hybridization with three-way DNA junction for in situ enhanced electronic detection of marine biotoxin based on isothermal cycling signal amplification strategy.

A new signal amplification strategy based on target-regulated DNA proximity hybridization (TRPH) reaction accompanying formation of three-way DNA junction was designed for electronic detection of Microcystin-LR (MC-LR used in this case), coupling with junction-induced isothermal cycling signal amplification. Initially, a sandwiched-type immunoreaction was carried out in a low-cost PCR tube between anti-MC-LR mAb1 antibody-labeled DNA1 (mAb1-DNA1) and anti-MC-LR mAb2-labeled DNA2 (mAb2-DNA2) in the presence of target to form a three-way DNA junction. Then, the junction could undergo an unbiased strand displacement reaction on an h-like DNA nanostructure-modified electrode (labeled with methylene blue redox tag on the short DNA strand), thereby resulting in the dissociation of methylene blue-labeled signal DNA from the electrode. The newly formed double-stranded DNA could be cleaved again by exonuclease III, and the released three-way DNA junction retriggered the strand-displacement reaction with h-like DNA nanostructures for junction recycling. During the strand-displacement reaction, numerous methylene blue-labeled DNA strands were far away from the electrode, thus decreasing the detectable electrochemical signal within the applied potentials. Under optimal conditions, the TRPH-based immunosensing system exhibited good electrochemical responses for detecting target MC-LR at a concentration as low as 1.0ngkg(-1) (1.0ppt). Additionally, the precision, reproducibility, specificity and method accuracy were also investigated with acceptable results.

Ultrasensitive and selective signal-on electrochemical DNA detection via exonuclease III catalysis and hybridization chain reaction amplification.

This work reported a novel, ultrasensitive, and selective platform for electrochemical detection of DNA, employing an integration of exonuclease III (Exo-III) assisted target recycling and hybridization chain reaction (HCR) for the dual signal amplification strategy. The hairpin capture probe DNA (C-DNA) with an Exo-III 3' overhang end was self-assembled on a gold electrode. In the presence of target DNA (T-DNA), C-DNA hybridized with the T-DNA to form a duplex region, exposing its 5' complementary sequence (initiator). Exo-III was applied to selectively digest duplex region from its 3-hydroxyl termini until the duplex was fully consumed, leaving the remnant initiator. The intact T-DNA spontaneously dissociated from the structure and then initiated the next hybridization process as a result of catalysis of the Exo-III. HCR event was triggered by the initiator and two hairpin helper signal probes labeled with methylene blue, facilitating the polymerization of oligonucleotides into a long nicked dsDNA molecule. The numerous exposed remnant initiators can trigger more HCR events. Because of integration of dual signal amplification and the specific HCR process reaction, the resultant sensor showed a high sensitivity for the detection of the target DNA in a linear range from 1.0 fM to 1.0 nM, and a detection limit as low as 0.2 fM. The proposed dual signal amplification strategy provides a powerful tool for detecting different sequences of target DNA by changing the sequence of capture probe and signal probes, holding a great potential for early diagnosis in gene-related diseases.

Gelatin methacrylate (GelMA) mediated electrochemical DNA biosensor for DNA hybridization.

In this study, an electrochemical biosensor system for the detection of DNA hybridization by using gelatin methacrylate (GelMA) modified electrodes was developed. Electrochemical behavior of GelMA modified Pencil Graphite Electrode (PGE) that serve as a functional platform was investigated by using Cyclic Voltammetry (CV) and Electrochemical Impedance Spectroscopy (EIS) and compared with those of the bare PGE. Hybridization was achieved in solution phase and guanine oxidation signal changes were evaluated. The decrease in the guanine oxidation peak currents at around +1.0 V was used as an indicator for the DNA hybridization. Also, more interestingly GelMA intrinsic oxidation peaks at around +0.7 V changed substantially by immobilization of different oligonucleotides such as probe, hybrid and control sequences to the electrode surface. It is the first study of using GelMA as a part of an electrochemical biosensor system. The results are very promising in terms of using GelMA as a new DNA hybridization indicator. Additionally, GelMA modified electrodes could be useful for detecting ultra low quantity of oligonucleotides by providing mechanical support to the bio-recognition layer. The detection limit of this method is at present 10(-12)mol. Signal suppressions were increased from 50% to 93% for hybrid with using GelMA when it was compared to bare electrode which facilitates the hybridization detection.

Whole-mount in situ hybridization to assess advancement of development and embryo morphology.

Whole-mount in situ hybridization (WISH) is widely used to visualize the site and dynamics of gene expression during embryonic development. Various methods of probe labeling and hybridization detection are available nowadays. Meanwhile the technique was adapted to be used on many different species and has evolved from a manual to a larger scale and automated procedure. Standardized automated protocols improve the chance to compare different experimental settings reliably. The high resolution of this method is ideally suited for examination of manipulated (e.g., cloned) embryos often displaying subtle changes only. Embedding and sectioning of in situ hybridized specimen further enhance the detailed examination of their gene expression and morphology.