Probe Hybridization Research Articles

Development of female specific SCAR marker in spine gourd (Momordica dioica Roxb. Ex. Wild) and its validation

Momordica dioica is a dioecious vegetable crop belonging to the family Cucurbitaceae. Fifty RAPD primers were used to analyze the male and female DNA bulks consisting of six females and six male genotypes of Momordica dioica respectively and 23 of them have differentiated the reproductive DNA bulks. One of the primers, OPB07, amplified a female reproductive bulk specific fragment of 326 bp (OPB07326) and was also present exclusively in all female genotypes used to prepare the reproductive bulk. This unique fragment OPB07326 was cloned and sequenced and was developed into female specific SCAR marker MdFSOPB07255. This SCAR marker has also amplified the unique female-specific fragment of 255 bp in all the six female genotypes used for the preparation of the bulk. The existence of this female-specific fragment in M. dioca genome was also affirmed by southern hybridization of DIG-labeled probe of OPB07326 with EcoRI restricted profile of all the six female genotypes. Further, the MdFSOPB07255 marker was successfully employed to identify eight female genotypes out of ten M. dioica genotypes with unknown reproductive feature. Thus, MdFSOPB07255 could be utilized as an efficient and reliable DNA marker for early differentiation of reproductive type at the sapling stage.

Abstract 1137: Spatial expression profile of mucinous cystic neoplasm indicates a divergent malignant marker patterns in ductal and stromal regions

Abstract Mucinous cystic pancreatic neoplasms (MCPN), initially characterized as benign, present a unique challenge, with the five-year survival rate dropping from 100% to 57% if malignancy occurs. In this study, we using spatial genomic expression analysis of MCPN, and focus on the correlations between marker expression patterns in stroma and ductal region. Tissues obtained from surgical samples of a 69-year-old patient with a pancreatic head cystic mass and a history of pancreatitis (staged as T3N1Mx) were embedded in paraffin. These tissues were then sectioned, collected onto subbed slides, and subjected to probe hybridization, ligation, and extraction onto Visum slides following the manufacturer's instructions (10X Genomics). Genomic expression was extracted using established protocols, processed through SpaceRanger, and further visualized in Loupe Browser (10X Genomics). A comparison was conducted between the ductal and stromal regions, both with and without cystic involvement. Our study reveals a significant 5.1 log2 fold increase in MUC5AC expression in the ductal region, regardless of cystic involvement, suggesting implications for apoptosis regression and cadherin-dependent cell adhesion in MCPN progression. Further analysis indicates a significantly higher expression of TMSB4X exclusively in the ductal region, extending into the surrounding stroma, potentially stimulating MCPN progression by fostering pro-inflammatory cytokine environments. The novel marker IGKC, indicating favorable prognosis and PDAC treatment efficacy, shows high expression in the stroma (1.8 log2 fold higher). However, around the duct with cystic involvement, its expression is notably reduced (-1.7 log2 fold lower), suggesting pathways that may contribute to MCPN's progression towards a more invasive PDAC phenotype. Additionally, our study reveals a 1.9 log2 fold elevated expression of FN1, a biomarker associated with poor outcomes in PDAC patients, in the stroma around the cyst, hinting at a possible pathogenic role of FN1 in cell adhesion, migration, and progression of MCPN to PDAC. Co-expression of FN1 with COMP and TIMP3 within MCPN suggests a concerted effort in the microenvironment, influencing tissue development, maintenance, and extracellular matrix remodeling. Co-localization of FN1 with COMP and TIMP3, particularly within degenerating acinar cells, hints at a potential mechanism in the intricate progression of MCPN to PDAC. These findings contribute to a deeper understanding of the molecular landscape of MCPN, offering potential avenues for targeted interventions to thwart its progression to PDAC, guiding novel diagnostic and therapeutic strategies for pancreatic neoplasms. Further expression data will be presented. Citation Format: Arshia Ghodrati, Chirag S. Gopinath, Amin Parvizi, Manu Gnanamony, Lusine Demirkhanyan, Christopher S. Gondi. Spatial expression profile of mucinous cystic neoplasm indicates a divergent malignant marker patterns in ductal and stromal regions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1137.

Abstract 5565: Multiomic characterization of colorectal cancer using MICS technology reveals interaction of antigen presenting cancer associated fibroblasts and T cells

Abstract In solid tumors, the tumor microenvironment (TME) is composed of diverse cell types including cancer cells, immune cells, stromal cells, and other tissue specific cell types. The complex intercellular interactions that occur between these various cell populations determine cancer development and progression. Cancer-associated fibroblasts (CAFs) have been identified as key players in the TME, capable of promoting tumor cell growth and invasion, as well as manipulating immune responses. To better resolve potential subpopulations, spatial relationships, and signaling occurring between cell types within the TME, we performed same-section multiomic profiling of colorectal cancer (CRC) using the MACSima™ Imaging Cyclic Staining (MICS) technology. We combined a custom 40plex RNA panel with a panel of antibodies to characterize the drivers of tumorigenesis and the activation state of immune cells. FFPE CRC specimens were reviewed by a pathologist for clinical assessment and region of interest selection. Then, gene expression profiles were generated using RNAsky™ technology with each gene being detected via cyclic rounds of detection probe hybridization, image acquisition, and signal erasure. Subsequently, fluorescently labeled antibodies were applied to the same section following an equivalent acquisition process. Finally, multiomic clustering and cell population analyses were performed using MACS® iQ View software. The analysis revealed spatially separated subpopulations of CAFs. Functional characterization of cellular neighborhoods and the cell-to-cell interactions occurring within the TME showed that one of the CAF populations potentially promoted cancer cell growth while another subpopulation, resembling antigen-presenting CAFs (apCAFs), closely interacted with T cells in the TME. These findings will deepen our understanding of tumor progression in colorectal cancer and potentially other solid tumors. Citation Format: Emily Neil, Rebecca C. Hennessey, David Agorku, Dongju Park, Julia Femel, Michael DiBuono, Hanna Lafayette, Erica Lloyd, Hsinyi Lo, Alex Makrigiorgos, Shaina Lu, John Lee, Sameh Soliman, Dominic Mangiardi, Paurush Praveen, Philip Ströbel, Silvia Rüberg, Fabian Staubach, Ryan Hindman, Thomas Rothmann, Olaf Hardt, Hansueli Meyer, Tanya Wantenaar, Jinling Wang, Werner Müller, Robert Pinard, Andreas Bosio. Multiomic characterization of colorectal cancer using MICS technology reveals interaction of antigen presenting cancer associated fibroblasts and T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5565.

A sensitive one-pot ROA assay for rapid miRNA detection

MicroRNAs (miRNAs) and short RNA fragments (18–25 nt) are crucial biomarkers in biological research and disease diagnostics. However, their accurate and rapid detection remains a challenge, largely due to their low abundance, short length, and sequence similarities. In this study, we report on a highly sensitive, one-step RNA O-circle amplification (ROA) assay for rapid and accurate miRNA detection. The ROA assay commences with the hybridization of a circular probe with the test RNA, followed by a linear rolling circle amplification (RCA) using dUTP. This amplification process is facilitated by U-nick reactions, which lead to an exponential amplification for readout. Under optimized conditions, assays can be completed within an hour, producing an amplification yield up to the microgram level, with a detection limit as low as 0.15 fmol (6 pM). Notably, the ROA assay requires only one step, and the results can be easily read visually, making it user-friendly. This ROA assay has proven effective in detecting various miRNAs and phage ssRNA. Overall, the ROA assay offers a user-friendly, rapid, and accurate solution for miRNA detection.

Transient states during the annealing of mismatched and bulged oligonucleotides.

Oligonucleotide hybridization is crucial in various biological, prebiotic and nanotechnological processes, including gene regulation, non-enzymatic primer extension and DNA nanodevice assembly. Although extensive research has focused on the thermodynamics and kinetics of nucleic acid hybridization, the behavior of complex mixtures and the outcome of competition for target binding remain less well understood. In this study, we investigate the impact of mismatches and bulges in a 12bp DNA or RNA duplex on its association (kon) and dissociation (koff) kinetics. We find that such defects have relatively small effects on the association kinetics, while the dissociation kinetics vary in a position-dependent manner by up to 6 orders of magnitude. Building upon this observation, we explored a competition scenario involving multiple oligonucleotides, and observed a transient low specificity of probe hybridization to fully versus partially complementary targets in solution. We characterize these long-lived metastable states and their evolution toward equilibrium, and show that sufficiently long-lived mis-paired duplexes can serve as substrates for prebiotically relevant chemical copying reactions. Our results suggest that transient low accuracy states may spontaneously emerge within all complex nucleic acid systems comprising a large enough number of competing strands, with potential repercussions for gene regulation in the realm of modern biology and the prebiotic preservation of genetic information.

Distribution of the Prevalence of Human Leukocyte Antigen (HLA)-B*57:01 Positivity in HIV-1 Infected Individuals and Its Effects on Treatment: Türkiye Map-Buhasder Working Group

Human immunodeficiency virus (HIV)/acquired immundeficiency syndrome (AIDS) is a critical global public health problem that significantly affects both life expectancy and the overall quality of life of individuals in all age groups. The landscape of HIV infection has changed significantly in recent years due to the introduction of effective combination antiretroviral therapies (ART). A key component of first-line ART regimens for HIV treatment is abacavir, a nucleoside HIV reverse transcriptase inhibitor. Although abacavir is effective in suppressing viral replication and managing disease, its clinical utility is overshadowed by the potential for life-threatening hypersensitivity reactions in HLA-B*57:01-positive patients. In our country, local data obtained from various centers regarding the prevalence of HLA-B*57:01 in HIV-1-infected patients are available. In this study, it was aimed to determine the prevalence of the HLA-B*57:01 genotype in HIV-infected patients who were followed up and treated in many regions of our country. This retrospective study consists of the data of the patients aged 18 years and over diagnosed with HIV-1 infection between 01.01.2019 and 31.07.2022. Age, gender, place of birth, mode of transmission of the disease, death status, CD4+ T cell count and HIV RNA levels at the first clinical presentation, HLA-B*57:01 positivity, and the method used, clinical stage of the disease, virological response time with the treatment they received were recorded from the patient files. Data were collected from 16 centers and each center used different methods to detect HLA-B*57:01. These methods were sequence-specific oligonucleotide probe hybridization (SSOP), DNA sequence-based typing (SBT), single-specific primer-polymerase chain reaction (SSP-PCR), allele-specific PCR (AS-PCR) and quantitative PCR (Q-PCR). A total of 608 HIV-infected individuals, 523 males (86%) and 85 females (14%), were included in the study. The mean age of the patients was 36.9 ± 11.9 (18-73) years. The prevalence of HLA-B*57:01 allele was found to be 3.6% (22 patients). The number of CD4+ T lymphocytes in HLA-B*57:01 allele-positive patients was > 500/ mm3 in 10 patients (45.5%), while the number of CD4+ T lymphocytes in HLA-B*57:01 negative patients was > 500/mm3 in 216 patients (36.9%) (p> 0.05). Viral load at the time of diagnosis was found to be lower in patients with positive HLA-B*57:01 allele but it was not statistically significant (p> 0.05). Although different treatment algorithms were used in the centers following the patients, it was observed that the duration of virological response was shorter in HLA-B*57:01 positive patients (p= 0.006). Although the presence of the HLA-B*57:01 allele has a negative impact due to its association with hypersensitivity, it is likely to continue to attract interest due to its association with slower progression of HIV infection and reduced risk of developing AIDS. In addition, although the answer to the question of whether it is cost-effective to screen patients for HLA-B*57:01 before starting an abacavir-containing ART regimen for the treatment of HIV infection is being sought, it seems that HIV treatment guidelines will continue to recommend screening to identify patients at risk in this regard.

SmFISH for Plants.

Single-molecule fluorescence in situ hybridization (smFISH) is a powerful method for the visualization and quantification of individual RNA molecules within intact cells. With its ability to probe gene expression at the single cell and single-molecule level, the technique offers valuable insights into cellular processes and cell-to-cell heterogeneity. Although widely used in the animal field, its use in plants has been limited. Here, we present an experimental smFISH workflow that allows researchers to overcome hybridization and imaging challenges in plants, including sample preparation, probe hybridization, and signal detection. Overall, this protocol holds great promise for unraveling the intricacies of gene expression regulation and RNA dynamics at the single-molecule level in whole plants.

Highly Multiplexed and Simultaneous Characterization of Protein and RNA in Single Cells by Flow or Mass Cytometry Platforms Using Proximity Ligation Assay for RNA.

In situ hybridization of oligonucleotide probes to intracellular RNA allows quantification of predefined gene transcripts within millions of single cells using cytometry platforms. Previous methods have been hindered by the number of RNA that can be analyzed simultaneously. Here we describe a method called proximity ligation assay for RNA (PLAYR) that permits highly multiplexed RNA analysis that can be combined with antibody staining. Potentially any number of RNA combined with antigen can be analyzed together, being limited only by the number of analytes that can be measured simultaneously.

Liquid and Solid Hybridization Methods to Detect RNAs.

Northern blotting (NB) has been a long-standing method for RNA detection. However, its labor-intensive nature, reliance on high-quality RNA, and use of radioactivity have diminished its appeal over time. Nevertheless, the emergence of microRNAs (miRNAs) has reignited the demand for sensitive and quantitative NB techniques. We have recently developed cost-effective and rapid protocols for RNA detection using solid and liquid hybridization (LH) techniques which exhibit high sensitivity without the need for radioactive or specialized reagents like locked nucleic acid (LNA) probes. Our assays incorporate biotinylated probes and improved techniques for probe hybridization, transfer, cross-linking, and signal enhancement. We demonstrate that while NB is sensitive in detecting mRNAs and small RNAs, our LH protocol efficiently detects these as well as miRNAs at lower amounts of RNA, achieving highersensitivity comparable to radiolabeled probes. Compared to NB, LH offers benefits of speed, sensitivity, and specificity in detecting mRNAs, small RNAs, and miRNAs.

Single Cell Transcriptomics Define Cell Heterogeneity in Limbal/Corneal Cells Relevant to Nerve Regeneration

Balanced interplay of cell-type specific transcriptomic regulation is critical for nerve regeneration in the central and peripheral nervous system. As a model, we use the cornea, where we recently discovered the RvD6-isomer and its function as a regulator of nerve regeneration, critical for dry eye in aging. Single-RNA-sequencing (snRNASeq) and spatial transcriptomics were used to interrogate cell populations and gene expression in central cornea/limbus from male, 3-month, C57BL/6 wild-type (WT) and injured mice cornea. Isolated nuclei from cornea/limbus of WT mice (N=12) were processed by 10×Genomics pipeline Cellranger. Central mice corneas were damaged with 2mm trephine, and epithelium and anterior stroma removed with corneal rust ring remover (Algerbrush II). Two days post-injury, corneas were excised, formalin-fixed paraffin-embedded, and 5µm sections prepared for Visium CytAssist mediated probe hybridization, library construction, sequencing, data analysis using Spaceranger, data visualization and cell cluster annotation using Loupe Browser. Cell populations were distinguished by t-distributed stochastic neighbor embedding (t-SNE). RNAscope® assay was used to validate two genes, ALDH3A1 and SPARC.

In Situ Hybridization to Characterize Neurulation and Midbrain-Hindbrain Boundary Formation in Zebrafish.

Whole-mount in situ hybridization is cable to harness the inherent advantages of zebrafish as a model organism for developmental biology, particularly when visualizing the formation of the neural tube, specifically at the level of the midbrain-hindbrain boundary. The size and transparency of developing zebrafish embryos allow for the visualization of neural markers in vivo along the length of the developing zebrafish central nervous system. In practice, this technique is useful for examining defects in neurulation and midbrain-hindbrain boundary formation that may arise following gene manipulation, for example, CRISPR mutagenesis. This method describes the process of embryo collection and preparation, RNA probe transcription, probe hybridization in vivo, as well as the process of probe detection and visualization.

Clonal Proliferation Within Smooth Muscle Cells in Unstable Human Atherosclerotic Lesions.

Studies in humans and mice using the expression of an X-linked gene or lineage tracing, respectively, have suggested that clones of smooth muscle cells (SMCs) exist in human atherosclerotic lesions but are limited by either spatial resolution or translatability of the model. Phenotypic clonality can be detected by X-chromosome inactivation patterns. We investigated whether clones of SMCs exist in unstable human atheroma using RNA in situ hybridization (BaseScope) to identify a naturally occurring 24-nucleotide deletion in the 3'UTR of the X-linked BGN (biglycan) gene, a proteoglycan highly expressed by SMCs. BGN-specific BaseScope probes were designed to target the wild-type or deletion mRNA. Three different coronary artery plaque types (erosion, rupture, and adaptive intimal thickening) were selected from heterozygous females for the deletion BGN. Hybridization of target RNA-specific probes was used to visualize the spatial distribution of mutants. A clonality index was calculated from the percentage of each probe in each region of interest. Spatial transcriptomics were used to identify differentially expressed transcripts within clonal and nonclonal regions. Less than one-half of regions of interest in the intimal plaque were considered clonal with the mean percent regions of interest with clonality higher in the intimal plaque than in the media. This was consistent for all plaque types. The relationship of the dominant clone in the intimal plaque and media showed significant concordance. In comparison with the nonclonal lesions, the regions with SMC clonality had lower expression of genes encoding cell growth suppressors such as CD74, SERF-2 (small EDRK-rich factor 2), CTSB (cathepsin B), and HLA-DPA1 (major histocompatibility complex, class II, DP alpha 1), among others. Our novel approach to examine clonality suggests atherosclerosis is primarily a disease of polyclonally and to a lesser extent clonally expanded SMCs and may have implications for the development of antiatherosclerotic therapies.

SENP1-mediated SUMOylation of SIRT1 affects glioma development through the NF-κB pathway

Gliomas are the most common primary brain tumors in adults. Although they exist in different malignant stages, most gliomas are clinically challenging because of their infiltrative growth patterns and inherent relapse tendency with increased malignancy. Epigenetic alterations have been suggested to be an important factor for glioma genesis. Using mRNA probe hybridization, we identified SUMO-specific protease 1 (SENP1) as the most significantly upregulated SUMOylation regulator in glioma. Moreover, SENP1 was overexpressed in gliomas and predicted poor prognoses. Depletion of SENP1 reduced glioma cell activity, cycle arrest, and increased apoptotic activity. Mechanistically, SENP1 inhibited the protein expression of sirtuin 1 (SIRT1) through de-SUMOylation, and SIRT1 inhibited the activity of nuclear factor kappaB (NF-κB) by deacetylation. Rescue experiments revealed that downregulation of SIRT1 reversed the inhibitory effect of sh-SENP1 on glioma cell malignant phenotype, while downregulation of NF-κB reversed the activating effect of sh-SIRT1 on glioma cell malignant phenotype. Thus, SENP1-mediated de-SUMOylation of SIRT1 might be therapeutically important in gliomas.

Alternative molecular and genomic strategies to provide a rapid response to alerts concerning the introduction of new emerging SARS-CoV-2 variants: the Omicron alert.

During the COVID-19 pandemic, several SARS-CoV-2 variants of concern (VOCs) of particular relevance emerged. Early detection of VOCs entering a country is essential to control spread. The alert triggered by the first suspected case of the Omicron variant in Spain in a traveler arriving from South Africa in November 2021 provided a unique opportunity to evaluate four different methodological strategies tailored to rapid identification of Omicron. The different approaches were designed to respond to the different technical resources available in different settings. First, we used melting probes in RT-PCR to determine the presence of four Omicron signatures (K417N, E484A, P681H, and absence of L452R): three probes showed deviations in temperature (Tm) values relative to the reference codons (E484K-15.8°C, P681H-5.2°C, and L452R-7.2°C) and one maintained the reference value (K417N). The deviation in Tm of P681H suggested the presence of the characteristic Omicron N679K mutation in the probe hybridization region; these data pointed to the presence of Omicron alleles. Second, the presence of 29 of the 33 characteristic single nucleotide polymorphisms (SNPs) in the Omicron variant S-gene was identified by Sanger sequencing of nine amplicons. The final two strategies involved identification of 47 of the 50 non-synonymous and indel mutations attributed to Omicron by rapid nanopore whole genome sequencing (WGS) and by Illumina WGS technology. These strategies enabled us to pre-assign the first Omicron case in Spain with high certainty 2 h after receipt of RNA and to confirm it genomically 3 h later, so that the Public Health authorities could be rapidly notified. IMPORTANCE The study presents different experimental alternatives to identify new variants of concern (VOCs) of SARS-CoV-2 entering a certain population. Early detection of a new VOC is crucial for surveillance and control of spread. The objective is to provide laboratories with tools adapted to their resource capabilities that offer a sufficient level of resolution to rule out, confirm, or pre-assign the presence of a suspected VOC. The study describes four different techniques that were applied simultaneously to the first suspected Omicron case in Spain, highlighting the level of resolution and response time achieved in each case. These techniques are based on the detection of mutations in the S-gene of the virus that can easily adapt to potential emerging variants. The results of the study allow any laboratory to prepare for new alerts of SARS-CoV-2 VOCs.

Fluid shear stress induces a shift from glycolytic to amino acid pathway in human trophoblasts

BackgroundThe human placenta, a tissue with a lifespan limited to the period of pregnancy, is exposed to varying shear rates by maternal blood perfusion depending on the stage of development. In this study, we aimed to investigate the effects of fluidic shear stress on the human trophoblast transcriptome and metabolism.ResultsBased on a trophoblast cell line cultured in a fluidic flow system, changes caused by shear stress were analyzed and compared to static conditions. RNA sequencing and bioinformatics analysis revealed an altered transcriptome and enriched gene ontology terms associated with amino acid and mitochondrial metabolism. A decreased GLUT1 expression and reduced glucose uptake, together with downregulated expression of key glycolytic rate-limiting enzymes, hexokinase 2 and phosphofructokinase 1 was observed. Altered mitochondrial ATP levels and mass spectrometry data, suggested a shift in energy production from glycolysis towards mitochondrial oxidative phosphorylation. This shift in energy production could be supported by increased expression of glutamic-oxaloacetic transaminase variants in response to shear stress as well as under low glucose availability or after silencing of GLUT1. The shift towards amino acid metabolic pathways could be supported by significantly altered amino acid levels, like glutamic acid, cysteine and serine. Downregulation of GLUT1 and glycolytic rate-limiting enzymes, with concomitant upregulation of glutamic-oxaloacetic transaminase 2 was confirmed in first trimester placental explants cultured under fluidic flow. In contrast, high fluid shear stress decreased glutamic-oxaloacetic transaminase 2 expression in term placental explants when compared to low flow rates. Placental tissue from pregnancies with intrauterine growth restriction are exposed to high shear rates and showed also decreased glutamic-oxaloacetic transaminase 2, while GLUT1 was unchanged and glycolytic rate-limiting enzymes showed a trend to be upregulated. The results were generated by using qPCR, immunoblots, quantification of immunofluorescent pictures, padlock probe hybridization, mass spectrometry and FRET-based measurement.ConclusionOur study suggests that onset of uteroplacental blood flow is accompanied by a shift from a predominant glycolytic- to an alternative amino acid converting metabolism in the villous trophoblast. Rheological changes with excessive fluidic shear stress at the placental surface, may disrupt this alternative amino acid pathway in the syncytiotrophoblast and could contribute to intrauterine growth restriction.

Alternative probe hybridization buffers for target RNA depletion and viral sequence recovery in NGS for poultry samples

Non-targeted next generation sequencing (NGS) is widely applied to identify the diversity of pathogens in field samples. However, abundance of host RNA (especially rRNA) and other environmental nucleic acids can reduce the abundance of pathogen specific reads of interest, reduce depth of coverage and increase surveillance costs. We presently deplete chicken- and selected bacterial-specific rRNAs in poultry field RNA samples with complementary DNA probes in a commercially available probe hybridization buffer followed by digestion of the RNA:DNA hybrids with RNase H. Because the current buffer is an expensive special order reagent of proprietary composition, we tested in-house and other commercially available buffers and identified a viable alternative that yields equivalent host rRNA depletion and viral-specific reads in poultry samples as the current special order reagent but at a reduced cost.

STR genotyping by real-time PCR using QueSTR probes

QueSTR probes provide a feasible alternative for implementing forensic Short Tandem Repeat (STR) genotyping on lab-on-a-chip (LoC) devices. Unlike capillary electrophoresis (CE), which poses challenges for LoC implementation, QueSTR probes have been demonstrated to accurately genotype STRs in a hybridization curve assay after PCR amplification. In this study, we modified the QueSTR probe assay for use as a hydrolysis probe assay during qPCR. An asymmetric real-time PCR was performed with QueSTR probes and RNase H2 in the master mix, during which the fluorescence was recorded. Designed to hybridize with specific STR alleles, QueSTR probes contain a fluorescent dye and quencher molecule, enabling detection of probe hybridization through RNase H2-mediated cleavage of the RNA:DNA duplex, which releases the quencher. Matching probes yielded lower threshold cycle values and steeper incline of fluorescence curves compared to non-matching probes, indicating accurate genotype. The QueSTR qPCR assay was used to successfully genotype four CODIS core loci (D16S539, D7S820, TPOX, and TH01) in 12 samples, with one exception. The integration of amplification and detection in a single reaction supports the use of QueSTR probes for miniaturizing STR genotyping, thereby complementing CE-based analyses in centralized labs.

New Thiazole Orange Derivatives for Improved Fluorescence Signaling of DNA FIT Probes

AbstractForced intercalation probes (FIT probes) are nucleic acid probes in which an intercalator dye of the thiazole orange (TO) family serves as a surrogate nucleobase. Hybridization of FIT probes is accompanied by enhancements of fluorescence. Looking for ways to increase turn-on and brightness of fluorescence, we herein report the synthesis of new fluorogenic base surrogates. In total, nine different TO derivatives were introduced into FIT probes. Fluorescence measurements in six different sequences revealed that substitution at both the quinoline and the benzothiazole part affects fluorescence turn-on upon hybridization and brightness of probe–target duplexes. A TO derivative containing a tricyclic benzothiazole provided FIT probes signaling hybridization by up to 18.6-fold enhancement of fluorescence. Improved fluorescence quantum yields (Φds up to 0.53) and high extinction coefficients (ε518 up to 91000 M–1·cm–1) make this dye an interesting, and in some sequences superior, alternative to the canonical thiazole orange used previously in FIT probes.

Discrimination of monozygotic twins using mtDNA heteroplasmy through probe capture enrichment and massively parallel sequencing.

Differentiating between monozygotic (MZ) twins remains difficult because they have the same genetic makeup. Applying the traditional STR genotyping approach cannot differentiate one from the other. Heteroplasmy refers to the presence of two or more different mtDNA copies within a single cell and this phenomenon is common in humans. The levels of heteroplasmy cannot change dramatically during transmission in the female germ line but increase or decrease during germ-line transmission and in somatic tissues during life. As massively parallel sequencing (MPS) technology has advanced, it has shown the extraordinary quantity of mtDNA heteroplasmy in humans. In this study, a probe hybridization technique was used to obtain mtDNA and then MPS was performed with an average sequencing depth of above 4000. The results showed us that all ten pairs of MZ twins were clearly differentiated with the minor heteroplasmy threshold at 1.0%, 0.5%, and 0.1%, respectively. Finally, we used a probe that targeted mtDNA to boost sequencing depth without interfering with nuclear DNA and this technique can be used in forensic genetics to differentiate the MZ twins.

PHDs-seq: a large-scale phenotypic screening method for drug discovery through parallel multi-readout quantification

High-throughput phenotypic screening is a cornerstone of drug development and the main technical approach for stem cell research. However, simultaneous detection of activated core factors responsible for cell fate determination and accurate assessment of directional cell transition are difficult using conventional screening methods that focus on changes in only a few biomarkers. The PHDs-seq (Probe Hybridization based Drug screening by sequencing) platform was developed to evaluate compound function based on their transcriptional effects in a wide range of signature biomarkers. In this proof-of-concept demonstration, several sets of markers related to cell fate determination were profiled in adipocyte reprogramming from dermal fibroblasts. After validating the accuracy, sensitivity and reproducibility of PHDs-seq data in molecular and cellular assays, a panel of 128 signalling-related compounds was screened for the ability to induce reprogramming of keloid fibroblasts (KF) into adipocytes. Notably, the potent ATP-competitive VEGFR/PDGFR inhibitor compound, ABT869, was found to promote the transition from fibroblasts to adipocytes. This study highlights the power and accuracy of the PHDs-seq platform for high-throughput drug screening in stem cell research, and supports its use in basic explorations of the molecular mechanisms underlying disease development.