Branched DNA Technology Research Articles

Rapid and Reliable Protein-Free HBV DNA Extraction and Sensitive Branched DNA Southern Blot Assay.

HBV covalently closed circular DNA (cccDNA) plays an important role in the persistence of hepatitis B virus (HBV) infection by serving as the template for transcription of viral RNAs. To cure HBV infection, it is expected that cccDNA needs either to be eliminated or silenced. Hence, precise cccDNA quantification is essential. Sample preparation is crucial to specifically detect cccDNA. Southern blot is regarded as the "gold standard" for specific cccDNA detection but lacks sensitivity. Here, we describe a rapid and reliable modified kit-based, HBV protein-free DNA extraction method as well as a novel enhanced sensitivity Southern blot that uses branched DNA technology to detect HBV DNA in cell culture and liver tissue samples. It is useful for both HBV molecular biology and antiviral research.

Abstract 5631: Expanding tools for multiplex mRNA imaging in spatialomics through the ViewRNA tissue assay kits

Abstract Spatialomics is a rapidly growing field as it allows researchers to gain a deeper understanding of transcriptomes and corresponding protein expression profiles in cells within complex tissue microenvironments. One critical technology for spatialomics is in-situ hybridization (ISH) technology, which enables direct visualization and quantitation of nucleic acid in cells with single molecule resolution. The Invitrogen ViewRNA ISH assays incorporate branched DNA (bDNA) technology provides tools for interrogating multiple RNA transcripts at the same time, paving the way for improved spatialomics research. This technology is a powerful tool for spatialomics, giving insight into important mechanisms within cells and tissue. With researchers increasingly using spatialomic data in fields like neuroscience, immuno-oncology & single-cell analysis, these scientists will need the rapid and efficient detection of mRNA co-expression profiles that ViewRNA portfolio provides. In our newest offering, ViewRNA fluorescence tissue kits we extended our branched DNA technology, proprietary probe set design and signal amplification technology with Alexa Fluor probes offering a unique, robust, and sensitive in situ hybridization assay for RNA localization in fixed tissues. With fluorescence detection using Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 594, Alexa Fluor 647, and Alexa Fluor 750, these kits allow multiplexed detection and imaging paving the way for improved spatialomics. This technology lays the foundation for expanded fluorescent profiles to provide scientists additional tools to probe multiple transcripts per sample, allow broader spatialomics research. For Research Use Only. Not for use in diagnostic procedures. Citation Format: Nancie Mooney. Expanding tools for multiplex mRNA imaging in spatialomics through the ViewRNA tissue assay kits. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5631.

Parallel monitoring of RNA abundance, localization and compactness with correlative single molecule FISH on LR White embedded samples.

Single mRNA molecules are frequently detected by single molecule fluorescence in situ hybridization (smFISH) using branched DNA technology. While providing strong and background-reduced signals, the method is inefficient in detecting mRNAs within dense structures, in monitoring mRNA compactness and in quantifying abundant mRNAs. To overcome these limitations, we have hybridized slices of high pressure frozen, freeze-substituted and LR White embedded cells (LR White smFISH). mRNA detection is physically restricted to the surface of the resin. This enables single molecule detection of RNAs with accuracy comparable to RNA sequencing, irrespective of their abundance, while at the same time providing spatial information on RNA localization that can be complemented with immunofluorescence and electron microscopy, as well as array tomography. Moreover, LR White embedding restricts the number of available probe pair recognition sites for each mRNA to a small subset. As a consequence, differences in signal intensities between RNA populations reflect differences in RNA structures, and we show that the method can be employed to determine mRNA compactness. We apply the method to answer some outstanding questions related to trans-splicing, RNA granules and mitochondrial RNA editing in single-cellular trypanosomes and we show an example of differential gene expression in the metazoan Caenorhabditis elegans.

Single-Cell Analysis of Cytokine mRNA and Protein Expression by Flow Cytometry.

Understanding how immune cells respond to external stimuli such as pathogens or drugs is a key component of biomedical research. Critical to the immune response are the expression of cell-surface receptors and the secretion of cytokines, which are tightly regulated by gene expression and protein synthesis. Previously, cytokine mRNA expression levels have been measured from bulk analysis of heterogeneous or sorted cell populations, and the correlation between cytokine mRNA expression and protein levels using these techniques can be highly variable. Flow cytometry is used to monitor changes in cell-surface and intracellular proteins, but some proteins such as cytokines may be transient and difficult to measure. Thus, a flow cytometry method that can simultaneously measure cytokine mRNA and protein levels in single cells is a very powerful tool. We defined a flow cytometry method that combines the conventional measurement of T cell surface proteins (CD45, CD3, CD4, CD8) and intracellular cytokines (IL-2, INF-γ) with fluorescent in situ hybridization and branched DNA technology for amplification and detection of IL-2 and INF-γ mRNA transcripts in activated T cells. This method has been applied to frozen peripheral mononuclear blood cells (PBMCs) and frozen blood samples, making it applicable to clinical trial specimens that require shipment to the test site. In CD4+ cells from activated PBMCs, the concordance between mRNA and protein levels was 41% for IL-2 and 21% for and INF-γ. In CD8+ cells from activated PBMCs, the concordance was 15% for IL-2 and 32% for INF-γ. © 2020 by John Wiley & Sons, Inc. Basic Protocol: Detection of IL-2 and IFN-γ mRNA and protein expression in frozen PBMCs Alternate Protocol: Detection of IL-2 and IFN-γ mRNA and protein expression in frozen blood.

Development of QuantiDNA ALU Assay on Luminex Platform for Monitoring Adverse Reactions to Radiation Therapy

Radiation treatment is required by 70% of cancer patients, however there is currently no clinical method for determining the therapeutic response or radiation induced toxicity that can be used during a course of radiation therapy to personalize the dose for individual patients. We developed a plate-based QuaniDNA® assay utilizing branched DNA technology (SuperbDNA®) to measure plasma cell free DNA concentration as a marker for monitoring radiation induced toxicity. Utilizing the same branched DNA technology, herein we describe the development of a new QuantiDNA® ALU assay on Luminex platform. The assay employs DNA capture probes coupled to MagPlex™ Microspheres and SuperbDNA® signal amplification technology with biotin-labelled signaling probes which bind to Streptavidin phycoerythrin (SAPE) and the fluorescence signal can be detected by Luminex instruments such as MAGPIX®. The assay can be performed directly on patient plasma samples and can be readily automated. Compared with previous plate-based QuantiDNA® assay, the new assay has a better sensitivity with the lower limit of detection (LOD) being 0.05 ng/ml, which equals 1 pg human DNA given that 20 ul of samples were loaded. The limit of quantitation of the assay is 0.11 ng/mL (equivalent to 2.2 pg human DNA). The inter- and intra-assay coefficients of variance were <20% and <15%, respectively. Bacterial DNA, EDTA (10mM), hemoglobin (500mg/dL) and cholesterol (300mg/mL) do not interfere with the assay. Thirty plasma samples from patients with prostate cancer were tested and the levels of circulating DNA in plasma pre-radiotherapy was 11.6 ∼ 130.6 ng/ml with a median value of 32.2 ng/ml. This assay can be used both for research and clinical testing of plasma samples for patients undergoing radiation therapy for optimization and personalization of treatment.

Effect of lesion proximity on the regenerative response of long descending propriospinal neurons after spinal transection injury

BackgroundThe spinal cord is limited in its capacity to repair after damage caused by injury or disease. However, propriospinal (PS) neurons in the spinal cord have demonstrated a propensity for axonal regeneration after spinal cord injury. They can regrow and extend axonal projections to re-establish connections across a spinal lesion. We have previously reported differential reactions of two distinct PS neuronal populations—short thoracic propriospinal (TPS) and long descending propriospinal tract (LDPT) neurons—following a low thoracic (T10) spinal cord injury in a rat model. Immediately after injury, TPS neurons undergo a strong initial regenerative response, defined by the upregulation of transcripts to several growth factor receptors, and growth associated proteins. Many also initiate a strong apoptotic response, leading to cell death. LDPT neurons, on the other hand, show neither a regenerative nor an apoptotic response. They show either a lowered expression or no change in genes for a variety of growth associated proteins, and these neurons survive for at least 2 months post-axotomy. There are several potential explanations for this lack of cellular response for LDPT neurons, one of which is the distance of the LDPT cell body from the T10 lesion. In this study, we examined the molecular response of LDPT neurons to axotomy caused by a proximal spinal cord lesion.ResultsUtilizing laser capture microdissection and RNA quantification with branched DNA technology, we analyzed the change in gene expression in LDPT neurons following axotomy near their cell body. Expression patterns of 34 genes selected for their robust responses in TPS neurons were analyzed 3 days following a T2 spinal lesion. Our results show that after axonal injury nearer their cell bodies, there was a differential response of the same set of genes evaluated previously in TPS neurons after proximal axotomy, and LDPT neurons after distal axotomy (T10 spinal transection). The genetic response was much less robust than for TPS neurons after proximal axotomy, included both increased and decreased expression of certain genes, and did not suggest either a major regenerative or apoptotic response within the population of genes examined.ConclusionsThe data collectively demonstrate that the location of axotomy in relation to the soma of a neuron has a major effect on its ability to mount a regenerative response. However, the data also suggest that there are endogenous differences in the LDPT and TPS neuronal populations that affect their response to axotomy. These phenotypic differences may indicate that different or multiple therapies may be needed following spinal cord injury to stimulate maximal regeneration of all PS axons.

Quantifying Tissue-Specific Overexpression of FOXO in Drosophila via mRNA Fluorescence In Situ Hybridization Using Branched DNA Probe Technology.

Whilethe highly conserved FOXO transcription factors have been studied in Drosophila melanogaster for decades, the ability to accurately control and measure their tissue-specific expression is often cumbersome due to a lack of reagents and to limited, nonhomogeneous samples. The need for quantitation within a distinct cell type is particularly important because transcription factors must be expressed in specific amounts to perform their functions properly. However, the inherent heterogeneity of many samples can make evaluating cell-specific FOXO and/or FOXO load difficult. Here, we describe an extremely sensitive fluorescence in situ hybridization (FISH) approach for visualizing and quantifying multiple mRNAs with single-cell resolution in adult Drosophila cardiomyocytes. The procedure relies upon branched DNA technology, which allows several fluorescent molecules to label an individual transcript, drastically increasing the signal-to-noise ratio compared to other FISH assays. This protocol can be modified for use in various small animal models, tissue types, and for assorted nucleic acids.

The immune response of Mytilus edulis hemocytes exposed to Vibrio splendidus LGP32 strain: A transcriptomic attempt at identifying molecular actors

The marine mussel Mytilus edulis, tolerant to a wide range of environmental changes, combines a key role as a sentinel species for environmental monitoring programs and a significant economic importance. Mortality events caused by infective agents and parasites have not been described in mussels, which suggests an efficient immune system. This study aims at identifying the molecular mechanisms involved in the early immune responses M. edulis' hemocytes challenged with Vibrio splendidus LGP32 strain during 2, 4 and 6 h.A total of 149,296 assembled sequences has been annotated and compared to KEGG reference pathways. Several immune related sequences were identified such as Toll-Like receptors (TLRs), transcription factors, cytokines, protease inhibitors, stress proteins and sequences encoding for proteins involved in cell adhesion, phagocytosis, oxidative stress, apoptosis and autophagy.Differential gene expression clustered 10 different groups of transcripts according to kinetics of transcript occurrence. Sequences were assigned to biological process gene ontology categories. Sequences encoding for galectins, fibrinogen-related proteins, TLRs, MyD88, some antimicrobial peptides, lysosomal hydrolases, heat shock proteins and protease inhibitors, as well as proteins of oxidative stress and apoptosis were identified as differently regulated during the exposure to V. splendidus LGP32.The levels of candidate transcripts were quantified in M. edulis' hemocytes exposed to V. splendidus LGP32 and 7SHRW by using branched DNA technology. Transcripts encoding for inhibitor kappa B, inhibitor of apoptosis proteins, tumor protein D54, serine/threonine-proteine kinase SIK2 were identified as up-regulated in hemocytes exposed to both strains.

Abstract 2741: A novel branched DNA-based multiplex method for telomere analysis at the single-cell level by flow cytometry

Abstract Telomeres are protective structures at the ends of chromosomes, containing multiple repeats of the repetitive sequence (TTAGGG) in vertebrates. It undergoes progressive shortening during normal aging. Abnormalities in the regulation of telomere length are implicated in various diseases including cancers. Current methods for telomere analysis are tedious and lack the flexibility to allow identification of unique cell subsets in a heterogeneous sample. Here we describe a novel flow cytometric method for analyzing relative telomere length at the single-cell level based on the branched DNA technology. This method, named PrimeFlow Assay can be combined with antibody staining and mRNA detection enabling simultaneous analysis of telomere length as well as protein and RNA for a comprehensive view in any specific cell subset. With this method, we investigated various cell lines and primary cells of human and mouse origin. Our results indicate that transformed cell lines possess longer telomeres than primary cells. In human peripheral blood mononuclear cells, we observed that lymphocytes had longer telomeres compared to monocytes, consistent with previously published results by another flow cytometric method. These data demonstrate that the PrimeFlow assay is a valuable method for high-throughput analysis of relative telomere length that may be combined with the analysis of protein and gene targets and, thus, provides data at the single-cell level for a more complete understanding of sample heterogeneity. Citation Format: Steve Lai, Leslie Sias, Castle Funatake. A novel branched DNA-based multiplex method for telomere analysis at the single-cell level by flow cytometry. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2741.

Albumin expression distinguishes bile duct adenomas from metastatic adenocarcinoma.

Bile duct adenomas may be difficult to distinguish from metastatic carcinomas, particularly well-differentiated pancreatic ductal adenocarcinoma. Prior studies have evaluated the utility of various immunohistochemical markers, although these markers are notable for low sensitivity and/or specificity. The aim of this study was to investigate the utility of albumin and BRAFV600E expression in distinguishing between metastatic pancreatic adenocarcinoma and bile duct adenoma. We studied 26 bile duct adenomas, three bile duct hamartomas, and 158 pancreatic ductal adenocarcinomas. Branched-chain in-situ hybridization (bISH) for albumin was performed; bISH is based on the branched DNA technology, wherein signal amplification is achieved via a series of sequential steps. Additionally, BRAFV600E immunohistochemistry (IHC) was performed on a subset of cases. Twenty-three of 25 (92%) bile duct adenomas were positive for albumin; 18 (72%) showed diffuse staining, and five showed focal staining (20%), including two challenging examples. Two bile duct hamartomas also stained positively. All pancreatic adenocarcinomas were negative for albumin. Seven of 16 (44%) bile duct adenomas and five of 106 (5%) pancreatic ductal adenocarcinomas were positive for BRAFV600E by IHC. The sensitivity and specificity ofexpression of albumin, as detected by bISH, for distinguishing bile duct adenomas from metastatic pancreatic adenocarcinomas were 92% and 100%, respectively; the sensitivity and specificity of BRAFV600E IHC for distinguishing bile duct adenomas from metastatic pancreatic adenocarcinomas were 43.8% and 95.3%, respectively. Diagnostically challenging examples of bile duct adenoma may be distinguished from metastatic pancreatic adenocarcinoma by the use of albumin bISH.

Application of HPV E6/E7 mRNA detection in screening of moderately severe cervical intraepithelial neoplasia

Objective This study aimed to explore the clinical value of human papilloma virus (HPV) E6/E7 mRNA tests in identifying precancerous lesions of the uterine cervix- cervical intraepithelial neoplasia 2 or more CIN2+ (CINⅡand CINⅢ). Methods This study is a cross-sectional survey design, between December 2011 to December 2013.The specimens were collected from the First People's Hospital of Huizhou and the Third People's Hospital of Huizhou in Department of Obstetrics and Gynecology outpatient and inpatient of cervical disease suspected patients, with thin-prep cytologic test (TCT) and histopathological results as reference, detected 345 patients of exfoliated cervical epithelial cells by using the branched DNA (b-DNA) technology to evaluate the application value of high risk HPV E6 / E7mRNA in the clinical diagnosis of CIN.Using spss 19.0 software for data analysis. Results (1)Compared with TCT, the positive rate of E6/E7 mRNA in 325 samples were grading by cytology as follows: no intraepithelial lesion cells (NILM) 21.1% (40/190), atypical squamous cells (ASC) 38.5% (15/39 ), low-grade squamous intraepithelial lesions (LSIL) 76.9% (30/39), atypical squamous cells can not exclude high-grade intraepithelial lesions (ASC-H) (8/10), high-grade squamous intraepithelial lesions (HSIL) 72.3% (34/47), TCT grades and HPV E6/E7 mRNA positive rate showed linear association (χ2=67.654, P<0.01; r=0.497, P<0.01); and with HPV E6/E7 mRNA copy number was also relevant (r=0.511, P<0.01). (2) Compared with pathological results, the positive rate of E6/E7 mRNA in 164 women samples were grading by pathology as follows: with NILM was 27.8%(10/36), with CIN Ⅰ was 65.9%(29/44), with CIN Ⅱ was 80.6%(54/67), and with CINⅢ was 82.4%(14/17), pathological grades and HPV E6/E7 mRNA positive rate showed a linear correlation (χ2= 26.426, P<0.01; r=0.438, P<0.01); and the number of copies correlated with the increase of pathological grades too(r=0.543, P<0.01). (3) Screening effectiveness analysis results showed, the sensitivity of HPV E6 / E7mRNA was 84.6% while TCT was 47.7%. The sensitivity and specificity were 40.0% and 91.1% respectively when HPV E6/E7 mRNA and TCT processed as sequential detection test. The CIN2 + (CIN Ⅱ and CIN Ⅲ) best diagnostic critical point of 890.26 copies/ml, was established using receiver operating characteristic (ROC) curve. The sensitivity and specificity were 58.5% and 93.7%, respectively. Conclusions The sensitivity of HPV E6/E7 mRNA test is better than TCT, the specificity is high in HPV E6/E7 mRNA and TCT processed as sequential detection test. Using the optimal cut-off value of ROC curve to detect CIN2+ has high sensitivity and specificity, so the detection of HPV E6/E7 mRNA may have some clinical value in screening and risk assessment of precancerous lesions of the uterine cervix.(Chin J Lab Med, 2015, 38: 532-536) Key words: Cervical intraepithelial neoplasia; Papillomaviridae; Oncogene proteins, viral; Papillomavirus E7 proteins

Prognostic significance of the mRNA expression of ERCC1, RRM1, TUBB3 and TYMS genes in patients with non-small cell lung cancer.

The present study aimed to investigate the prognostic value of excision repair cross-complementing 1 (ERCC1), ribonucleotide reductase subunit M1 (RRM1), class III β-tubulin (TUBB3) and thymidylate synthase (TYMS) in patients with non-small cell lung cancer (NSCLC) receiving platinum-based adjuvant chemotherapy. The mRNA expression of these genes was assessed in 72 tumor tissue samples obtained following surgery, using multiplex branched-DNA technology. Subsequent to surgery, all 72 patients with NSCLC were treated with platinum-based chemotherapy. The expression of these five genes was analyzed and the correlation with clinical characteristics and patient survival was investigated. Among the 72 samples, the incidence rate of mRNA expression of ERCC1 was 38.9% (28/72), RRM1 was 55.6% (40/72), TUBB3 was 47.2% (34/72) and TYMS was 62.5% (45/72). The incidence rate of ERCC1 expression in adenocarcinoma (34.2%) was significantly lower than that in non-adenocarcinoma (44.1%; P<0.05). Furthermore, the incidence rates of TYMS and TUBB3 expression in the high-median differentiation tissue samples were significantly lower than those in the low differentiation tissue samples (P<0.05). When the correlation of gene expression and patient survival was analyzed, high expression of ERCC1, RRM1, TUBB3 or TYMS was found to be associated with poor prognosis (P<0.001, P=0.001, P=0.001 and P=0.001, respectively). ERCC1, RRM1, TUBB3 and TYMS are key factors involved in survival following surgical treatment in patients with NSCLC. The mRNA expression of these genes may have prognostic value for patients with NSCLC treated with platinum-based chemotherapy.

High-throughput detection of miRNAs and gene-specific mRNA at the single-cell level by flow cytometry.

Fluorescent in situ hybridization (FISH) is a method that uses fluorescent probes to detect specific nucleic acid sequences at the single cell level. Here we describe optimized protocols that exploit a highly sensitive FISH method based on branched DNA technology to detect mRNA and miRNA in human leukocytes. This technique can be multiplexed and combined with fluorescent antibody protein staining to addressa variety of questions in heterogeneous cell populations. We demonstrate antigen-specific upregulation of IFNγ and IL-2 mRNAs in HIV- and CMV-specific T cells. We show simultaneous detection of cytokine mRNA and corresponding protein in single cells. We apply this method to detect mRNAs for which flow antibodies against the corresponding proteins are poor or are not available. We use this technique to show modulation of a microRNA critical for T cell function, miR-155. We adapt this assay for simultaneous detection of mRNA and proteins by Image Stream technology.

Image-based transcriptomics in thousands of single human cells at single-molecule resolution

Fluorescence in situ hybridization (FISH) is widely used to obtain information about transcript copy number and subcellular localization in single cells. However, current approaches do not readily scale to the analysis of whole transcriptomes. Here we show that branched DNA technology combined with automated liquid handling, high-content imaging and quantitative image analysis allows highly reproducible quantification of transcript abundance in thousands of single cells at single-molecule resolution. In addition, it allows extraction of a multivariate feature set quantifying subcellular patterning and spatial properties of transcripts and their cell-to-cell variability. This has multiple implications for the functional interpretation of cell-to-cell variability in gene expression and enables the unbiased identification of functionally relevant in situ signatures of the transcriptome without the need for perturbations. Because this method can be incorporated in a wide variety of high-throughput image-based approaches, we expect it to be broadly applicable.

The pathogenic and vaccine strains of equine infectious anemia virus differentially induce cytokine and chemokine expression and apoptosis in macrophages

The attenuated equine infectious anemia virus (EIAV) vaccine was the first attenuated lentivirus vaccine to be used in a large-scale application and has been used to successfully control the spread of equine infectious anemia (EIA) in China. To better understand the potential role of cytokines in the pathogenesis of EIAV infection and resulting immune response, we used branched DNA technology to compare the mRNA expression levels of 12 cytokines and chemokines, including IL-1α, IL-1β, IL-4, IL-10, TNF-α, IFN-γ, IP-10, IL-8, MIP-1α, MIP-1β, MCP-1, and MCP-2, in equine monocyte-derived macrophages (eMDMs) infected with the EIAVDLV121 vaccine strain or the parental EIAVDLV34 pathogenic strain. Infection with EIAVDLV34 and EIAVDLV121 both caused changes in the mRNA levels of various cytokines and chemokines in eMDMs. In the early stage of infection with EIAVDLV34 (0–24h), the expression of the pro-inflammatory cytokines TNF-α and IL-1β were significantly up-regulated, while with EIAVDLV121, expression of the anti-inflammatory cytokine IL-4 was markedly up-regulated. The effects on the expression of other cytokines and chemokines were similar between these two strains of virus. During the first 4 days after infection, the expression level of IL-4 in cells infected with the pathogenic strain were significantly higher than that in cells infected with the vaccine strain, but the expression of IL-1α and IL-1β induced by the vaccine strain was significantly higher than that observed with the pathogenic strain. In addition, after 4 days of infection with the pathogenic strain, the expression levels of 5 chemokines, but not IP-10, were markedly increased in eMDMs. In contrast, the vaccine strain did not up-regulate these chemokines to this level. Contrary to our expectation, induced apoptosis in eMDMs infected with the vaccine strain was significantly higher than that infected with the pathogenic strain 4 days and 6 days after infection. Together, these results contribute to a greater understanding of the pathogenesis of EIAV and of the mechanisms by which the immune response is induced after EIAV infection.

Gene expression analysis of leprosy by using a multiplex branched DNA assay

Leprosy is caused by Mycobacterium leprae, and the global registered prevalence of leprosy at the beginning of 2009 stood at 213,036 cases. It has long been thought that leprosy has a strong genetic risk. Recently, we have identified significant associations (P < 1.00 × 10(-10)) between SNPs in the genes CCDC122, C13orf31, NOD2, TNFSF15, HLA-DR and RIPK2 and a trend towards an association (P = 5.10 × 10(-5)) with a SNP in LRRK2. Here, we investigated the expression of these seven genes in formalin-fixed, paraffin-embedded skin tissues of leprosy and matched normal tissues using branched DNA technology. This technology allows for direct measurement of targeted mRNA within cellular lysate using a 96-well plate format in a time frame compared to a reporter gene assay. The clear upregulation of all seven genes was found in leprosy tissues compared to normal tissues, which further supports our genome-wide association study results.

Drug Transporter Expression and Localization in Rat Nasal Respiratory and Olfactory Mucosa and Olfactory Bulb: Fig. 1.

Uptake of drugs and other xenobiotics from the nasal cavity and into either the brain or systemic circulation can occur through several different mechanisms, including paracellular transport and movement along primary olfactory nerve axons, which extend from the nasal cavity to the olfactory bulb of the brain. The present study was conducted to expand knowledge on a third means of uptake, namely the expression of drug transporters in the rat nasal epithelium. We used branched DNA technology to compare the level of expression of nine transporters [(equilibrative nucleoside transporters (ENT)1 and ENT2; organic cation transporter (OCT)1, 2, and 3; OCTN1; organic anion-transporting polypeptide (OATP)3; and multidrug resistance (MRP)1 and MRP4] in nasal respiratory mucosa, olfactory mucosa, and olfactory bulb to the level of expression of these transporters in the liver and kidney. Transporters with high expression in the nasal respiratory mucosa or olfactory tissues were immunolocalized by immunohistochemistry. ENT1 and ENT2 expression was relatively high in nasal epithelia and olfactory bulb, which may explain the uptake of intranasally administered nucleoside derivatives observed by other investigators. OATP3 immunoreactivity was high in olfactory epithelium and olfactory nerve bundles, which suggests that substrates transported by OATP3 may be candidates for intranasal administration.

Editorial Comment

In 2005, a molecular pathology group led by AM Chinnaiyan used a novel computational analysis of pre-existing microarray datasets and uncovered recurrent gene fusions involving the prostate-specific gene transmembrane protease, serine 2 (TMPRSS2) and members of the erythroblastosis virus E26 transforming sequence (ETS) family of transcription factors in prostate cancer (PCa). The most frequent partner in the fusion was the ERG gene located at locus 21q22.2. TMPRSS2-ERG fusions are identifiable by a variety of molecular platforms, including fluorescent in situ hybridization (FISH), RT-PCR, and quantitative real-time RT-PCR, in 40%-60% of the acinar PCa. Although the significance of the rearrangement in terms of patient outcome remains somewhat controversial, it is now clear that TMPRSS2-ERG fusion is an early and specific event in the oncogenesis of PCa, with potential utility as a biomarker for PCa screening and diagnosis. 1 Tomlins S.A. Rhodes D.R. Perner S. et al. Recurrent fusion of TMPRSS2 and ETS transcription factor genes in prostate cancer. Science. 2005; 310: 644-648 Crossref PubMed Scopus (3056) Google Scholar , 2 Morris D.S. Tomlins S.A. Montie J.E. et al. The discovery and application of gene fusions in prostate cancer. BJU Int. 2008; 102: 276-282 Crossref Scopus (40) Google Scholar Detection of TMPRSS2-ERG Fusion Gene Expression in Prostate Cancer Specimens by a Novel Assay Using Branched DNAUrologyVol. 74Issue 5PreviewTo develop a novel assay that uses branched DNA technology to measure TMPRSS2-ERG fusion, as genetic rearrangement of TMPRSS2 regulatory sequences and coding sequences of the ERG gene has been detected in nearly half of prostate cancers, but quantitative assays to detect such TMPRSS2-ERG gene fusion have been limited to real-time polymerase chain reaction (PCR) techniques that rely on reverse transcriptase-based amplification. Full-Text PDF ReplyUrologyVol. 74Issue 5PreviewThe editorial comment provides an important counterbalance to our report of a new method for detecting TMPRSS2-ERG gene rearrangement in prostate cancer. This gene rearrangement juxtaposes a prostate-specific promoter (TMPRSS2) with a transcription factor that is not normally expressed in the prostate (ERG) and leads to expression of high levels of ERG by prostate cells that normally would not express ERG. The ERG transcription factor, in turn, can lead to expression of a host of other genes that are not expressed in normal prostate epithelium. Full-Text PDF

Detection of TMPRSS2- ERG Fusion Gene Expression in Prostate Cancer Specimens by a Novel Assay Using Branched DNA

To develop a novel assay that uses branched DNA technology to measure TMPRSS2-ERG fusion, as genetic rearrangement of TMPRSS2 regulatory sequences and coding sequences of the ERG gene has been detected in nearly half of prostate cancers, but quantitative assays to detect such TMPRSS2-ERG gene fusion have been limited to real-time polymerase chain reaction (PCR) techniques that rely on reverse transcriptase-based amplification. Branched DNA probes were designed to detect TMPRSS2-ERG gene fusion in prostate cancer cell lines. Nonquantitative nested reverse transcription (RT)-PCR and fluorescence in situ hybridization (FISH) were used to ascertain TMPRSS2-ERG gene fusion status in prostate tissues. The branched DNA assay detected TMPRSS2-ERG gene fusion from less than 200 pg of prostate cancer RNA, whereas more than 600 pg of RNA was required for fusion gene detection by one step real-time RT-PCR. In evaluation of clinical prostatectomy specimens, the branched DNA assay showed a concordant detectable fusion signal in all 9 clinical samples that had fusion detected by nested RT-PCR or FISH. Moreover, branched DNA detected gene fusion in 2 of 16 prostate cancer tissue specimens that was not detected by FISH or nested RT-PCR. Our findings demonstrate a branched DNA assay that is effective for detection of TMPRSS2-ERG gene fusion in prostate cancer clinical specimens, thus providing an alternative method to ascertain TMPRSS2-ERG gene fusion in human prostate cancer tissue.

Three Patterns of Cytochrome P450 Gene Expression during Liver Maturation in Mice

The neonatal period of liver development is an often overlooked phase of development. For instance, ontogeny of xenobiotic-metabolizing enzymes can markedly affect biotransformation as the liver matures. To systematically examine the ontogenic gene expression patterns of cytochrome P450 genes (P450) in mice, the gene expression profiles of 19 xenobiotic-metabolizing P450 in Cyp1 to 4 families were determined. The mRNA levels in C57BL/6 mouse livers were quantified using branched DNA technology at the following ages: gestational day 17 (2 days before birth) and postnatal days 0, 1, 3, 5, 10, 15, 20, 30, and 45. Among the 13 P450 genes expressed in mouse livers, three distinct ontogenic expression patterns were identified by cluster analysis. Genes in group 1 (Cyp3a16 as well as 3a41b in male) were expressed in the perinatal period, but they were essentially nondetectable by 30 days of age. Genes in group 2 (Cyp2e1, 3a11, and 4a10 as well as 3a41b in female) quickly increased after birth and reached maximal expression levels by day 5. Genes in group 3 (Cyp1a2, 2a4, 2b10, 2c29, 2d22, 2f2, 3a13, and 3a25) were expressed at low levels until days 10 to 15, but they markedly increased at day 20 to a high and stable level. In conclusion, the developmental expression of P450 in mouse liver can be divided into three patterns, suggesting that different mechanisms are responsible for the expression of P450 during liver maturation.