Transcription-polymerase Chain Reaction Approach Research Articles

Enhanced Detection of Viral RNA Species Using FokI-Assisted Digestion of DNA Duplexes and DNA/RNA Hybrids.

The accurate detection of nucleic acids from certain biological pathogens is critical for the diagnosis of human diseases. However, amplified detection of RNA molecules from a complex sample by direct detection of RNA/DNA hybrids remains a challenge. Here, we show that type IIS endonuclease FokI is able to digest DNA duplexes and DNA/RNA hybrids when assisted by a dumbbell-like fluorescent sensing oligonucleotide. As proof of concept, we designed a battery of sensing oligonucleotides against specific regions of the SARS-CoV-2 genome and interrogated the role of FokI relaxation as a potential nicking enzyme for fluorescence signal amplification. FokI-assisted digestion of SARS-CoV-2 probes increases the detection signal of ssDNA and RNA molecules and decreases the limit of detection more than 3.5-fold as compared to conventional molecular beacon approaches. This cleavage reaction is highly specific to its target molecules, and no detection of other highly related B-coronaviruses was observed in the presence of complex RNA mixtures. In addition, the FokI-assisted reaction has a high multiplexing potential, as the combined detection of different viral RNAs, including different SARS-CoV-2 variants, was achieved in the presence of multiple combinations of fluorophores and sensing oligonucleotides. When combined with isothermal rolling circle amplification technologies, FokI-assisted digestion reduced the detection time of SARS-CoV-2 in COVID-19-positive human samples with adequate sensitivity and specificity compared to conventional reverse transcription polymerase chain reaction approaches, highlighting the potential of FokI-assisted signal amplification as a valuable sensing mechanism for the detection of human pathogens.

Differential expression of plasma microRNA in preeclamptic patients at delivery and 1 year postpartum

Preeclampsia (PE) is a hypertensive disorder of pregnancy characterized by widespread maternal endothelial dysfunction. Although clinical signs subside following delivery, long-term risks associated with PE include hypertension, stroke, and cardiovascular disease. MicroRNAs (miRNAs) are emerging as critical regulators of biological function, and while alterations to the miRNAome have been described in the context of pregnancy and PE, the postpartum implications of PE on miRNA expression is unknown. The goal of this study was to characterize circulating miRNA profiles at the time of delivery and at 1 year postpartum for women who did and did not develop PE. Using a targeted reverse transcription polymerase chain reaction approach, selected miRNAs putatively involved in the pathophysiology of PE were examined in 17 normotensive control and 13 PE maternal plasma samples at the time of delivery and 1 year postpartum. Ingenuity Pathway Analysis was used to map putative messenger RNA targets of differentially expressed miRNA to global molecular networks based on gene function. Significant increases (P < .05) in 7 miRNAs with antiangiogenic, inflammatory, and apoptotic functions (miR-98-5p, miR-222-3p, miR-210-3p, miR-155-5p, miR-296-3p, miR-181a-5p, miR-29b-3p) were evident in maternal plasma at the time of severe PE compared to time-matched controls. Plasma samples from individuals who developed mild PE exhibited no changes compared to control samples for the subset of miRNAs analyzed here. Differential expression of plasma miRNA at the time of delivery for women with PE were largely resolved at 1 year postpartum, and reduced expression of only miR-221-3p (P < .05) was evident. Network analysis of putative targets of differentially regulated miRNA identified 11 interacting networks with enrichment for proteins involved in cardiovascular disease, organ system development and function, and cell signaling and interaction. The systemic effect of PE on maternal systems is evident in the circulating miRNAome with substantial alterations in miRNA expression in women who develop severe PE. In addition we provide novel evidence of disruption to miR-221 expression 1 year postpartum following a pregnancy complicated by PE compared to normotensive time-matched controls, which may allude to persistent inflammation in these women after delivery.

Investigation of osteosarcoma genomics and its impact on targeted therapy: an international collaboration to conquer human osteosarcoma

Osteosarcoma is a genetically unstable malignancy that most frequently occurs in children and young adults. The lack of progress in managing this devastating disease in the clinic has prompted international researchers to collaborate to profile key genomic alterations that define osteosarcoma. A team of researchers and clinicians from China, Finland, and the United States investigated human osteosarcoma by integrating transcriptome sequencing (RNA-seq), high-density genome-wide array comparative genomic hybridization (aCGH), fluorescence in situ hybridization (FISH), reverse transcription-polymerase chain reaction (RT-PCR), Sanger sequencing, cell culture, and molecular biological approaches. Systematic analysis of genetic/genomic alterations and further functional studies have led to several important findings, including novel rearrangement hotspots, osteosarcoma-specific LRP1-SNRNP25 and KCNMB4-CCND3 fusion genes, VEGF and Wnt signaling pathway alterations, deletion of the WWOX gene, and amplification of the APEX1 and RUNX2 genes. Importantly, these genetic events associate significantly with pathogenesis, prognosis, progression, and therapeutic activity in osteosarcoma, suggesting their potential impact on improved managements of human osteosarcoma. This international initiative provides opportunities for developing new treatment modalities to conquer osteosarcoma.

Gene Expression Profiling Reveals New Potential Players of Gonad Differentiation in the Chicken Embryo

BackgroundIn birds as in mammals, a genetic switch determines whether the undifferentiated gonad develops into an ovary or a testis. However, understanding of the molecular pathway(s) involved in gonad differentiation is still incomplete.Methodology/Principal FindingsWith the aim of improving characterization of the molecular pathway(s) involved in gonad differentiation in the chicken embryo, we developed a large scale real time reverse transcription polymerase chain reaction approach on 110 selected genes for evaluation of their expression profiles during chicken gonad differentiation between days 5.5 and 19 of incubation. Hierarchical clustering analysis of the resulting datasets discriminated gene clusters expressed preferentially in the ovary or the testis, and/or at early or later periods of embryonic gonad development. Fitting a linear model and testing the comparisons of interest allowed the identification of new potential actors of gonad differentiation, such as Z-linked ADAMTS12, LOC427192 (corresponding to NIM1 protein) and CFC1, that are upregulated in the developing testis, and BMP3 and Z-linked ADAMTSL1, that are preferentially expressed in the developing ovary. Interestingly, the expression patterns of several members of the transforming growth factor β family were sexually dimorphic, with inhibin subunits upregulated in the testis, and bone morphogenetic protein subfamily members including BMP2, BMP3, BMP4 and BMP7, upregulated in the ovary. This study also highlighted several genes displaying asymmetric expression profiles such as GREM1 and BMP3 that are potentially involved in different aspects of gonad left-right asymmetry.Conclusion/SignificanceThis study supports the overall conservation of vertebrate sex differentiation pathways but also reveals some particular feature of gene expression patterns during gonad development in the chicken. In particular, our study revealed new candidate genes which may be potential actors of chicken gonad differentiation and provides evidence of the preferential expression of BMPs in the developing ovary and Inhibin/Activin subunits in the developing testis.

Differential oxidative stress gene expression profile in mouse brain after proton exposure

Radiation is known to potentially interfere with cellular functions at all levels of cell organization. The radiation-induced stress response is very complex and involves altered expression of many genes. Identification of specific genes may allow the determination of pathways important in radiation responses. Although several radiation-related research have been studied extensively, the molecular and cellular processes affected by proton exposure remain poorly understood. Our earlier reports have shown that proton radiation induces reactive oxygen species (ROS) formation and lipid peroxidation and inhibits antioxidants, superoxide dismutase, and glutathione. Therefore, in this present study, we used quantitative real-time reverse transcription polymerase chain reaction approach and showed the modulation of several genes including oxidative stress, antioxidants defense mechanism, ROS metabolism, and oxygen transporters related genes expression in 2-Gy proton-exposed mouse brain. Literature evidences suggest that change in oxidants and antioxidants levels induce DNA damage, followed by cell death. In conclusion, changes in the gene profile of mouse brain after proton irradiation are complex and the exposed cells might undergo programmed cell death through alteration of genes responsible for oxidative stress signaling mechanism.

Receptor protein tyrosine phosphatases are expressed by cycling retinal progenitor cells and involved in neuronal development of mouse retina

Receptor protein tyrosine phosphatases (RPTPs) appear to coordinate many aspects of neural development, including cell proliferation, migration and differentiation. Here we investigated potential roles of RPTPs in the developing mouse retina. Using a degenerate oligonucleotide-based reverse transcription polymerase chain reaction approach, we identified 11 different RPTPs in the retina at embryonic stage 13 (E13). Subsequently, the expression patterns of RPTPκ, RPTPJ, RPTPRR, RPTPσ, RPTPε and RPTPγ in the retina from embryonic stages to adult were analyzed in detail using quantitative real-time-PCR, in situ hybridization, immunohistochemistry and Western blotting. At E13, all six RPTPs are expressed in actively cycling retinal progenitor cells and postmitotic newborn retinal neurons. With ongoing maturation, RPTPκ, RPTPJ, RPTPRR, RPTPσ, RPTPε and RPTPγ display a different spatiotemporal regulation of mRNAs and proteins in the pre- and postnatal retina. Finally, in adulthood these six RPTPs localize to distinct cellular compartments of multiple retinal neurons. Additional studies in RPTPγ −/− and RPTPβ/ζ −/− (also known as PTPRZ1, RPTPβ or RPTPζ) mice at postnatal stage P1 reveal no apparent differences in retinal laminar organization or in the expression pattern of specific retinal cell-type markers when compared with wild type. However, in RPTPβ/ζ −/− retinas, immunoreactivity of vimentin, a marker of Müller glial cells, is selectively reduced and the morphology of vimentin-immunoreactive radial processes of Müller cells is considerably disturbed. Our results suggest distinct roles of RPTPs in cell proliferation and establishing phenotypes of different retinal cells during retinogenesis as well as later in the maintenance of mature retina.

Identification of acute myeloid leukaemia associated microRNA expression patterns

MicroRNAs (miRNAs) play an important role in cellular differentiation and cancer pathogenesis. This study analysed the expression of 154 human miRNAs in acute myeloid leukaemia (AML) and control samples using a stem-loop real-time reverse transcription polymerase chain reaction approach. Global patterns of miRNA expression in AML, normal bone marrow (NBM) and CD34(+) progenitor cells allowed correct class predictions similar to whole genome microarray expression analyses that were performed at the same time. At single miRNA species level, MIRN23B was repressed in AML specimens compared to NBM and purified CD34(+) haematopoietic progenitor cells. In contrast, the MIRN221/MIRN222 cluster and MIRN34A were expressed at significantly higher levels in AML blasts. Patients with high MIRN221/MIRN222 expression showed low levels of KIT RNA and protein expression but the correlation between kit protein and KIT mRNA was significantly stronger than the correlation of either one with MIRN221/MIRN222. A global analysis between miRNA expression levels and mRNA expression of predicted target genes revealed only weak associations in the majority of miRNA species. Nonetheless, the presence of two or more miRNA binding sites within the mRNA was usually associated with a decrease in mRNA levels. Taken together, these findings provide evidence that specific miRNA expression patterns exist in AML.

Piezo-power microdissection of mature human dental tissue

Isolation of sufficient quantities of pure populations of odontoblasts from healthy and diseased teeth will facilitate our understanding of dentinogenesis during development and repair. Here we describe a novel Piezo-power microdissection (PPMD) technique for the isolation of pure populations of odontoblasts and pulpal tissue from formalin-fixed, paraffin-embedded, mature, healthy and carious human teeth. Odontoblasts and pulpal tissue gene expression were subsequently studied in ribonucleic acid isolated from PPMD preparations using a semi-quantitative reverse transcription polymerase chain reaction approach. Data confirmed that the genes for dentine sialophosphoprotein and Nestin are preferentially expressed in odontoblasts, whilst the genes for both collagen-1α and collagen-3α were expressed preferentially in pulpal tissue, particularly in carious samples. PPMD provides a novel and powerful approach to isolate pure populations of dental tissues and cells from fixed specimens for subsequent downstream molecular analyses.

Divergent evolution of flavonoid 2-oxoglutarate-dependent dioxygenases in parsley

Flavone synthases (FNSs) catalyze the oxidation of flavanones to flavones, i.e. the formation of apigenin from (2 S)-naringenin. While many plants express a microsomal-type FNS II, the soluble FNS I appears to be confined to a few species of the Apiaceae and was cloned recently from parsley plants. FNS I belongs to the Fe II/2-oxoglutarate-dependent dioxygenases characterized by short conserved sequence elements for cofactor binding, and its evolutionary context and mode of action are under investigation. Using a homology-based reverse transcription polymerase chain reaction approach, two additional flavonoid-specific dioxygenases were cloned from immature parsley leaflets, which were identified as flavanone 3β-hydroxylase (FHT) and flavonol synthase (FLS) after expression in yeast cells. Sequence alignments revealed marginal differences among the parsley FNS I and FHT polypeptides of only 6%, while much less identity (about 29%) was observed with the parsley FLS. Analogous to FNS I, FLS oxidizes the flavonoid γ-pyrone by introducing a C2, C3 double bond, and (2 R,3 S)-dihydrokaempferol ( cis-dihydrokaempferol) was proposed recently as the most likely intermediate in both FNS I and FLS catalysis. Incubation of either FNS I or FLS with cis-dihydrokaempferol exclusively produced kaempferol and confirmed the assumption that flavonol formation occurs via hydroxylation at C3 followed by dehydratation. However, the lack of apigenin in these incubations ruled out cis-dihydrokaempferol as a free intermediate in FNS I catalysis. Furthermore, neither (+)- trans-dihydrokaempferol nor unnatural (−)- trans-dihydrokaempferol and 2-hydroxynaringenin served as a substrate for FNS I. Overall, the data suggest that FNS I has evolved uniquely in some Apiaceae as a paraphyletic gene from FHT, irrespective of the fact that FNS I and FLS catalyze equivalent desaturation reactions.

MDR1 modulators improve the chemotherapy response of human hepatoblastoma to doxorubicin in vitro

Purpose: P-glycoprotein, a membrane efflux pump encoded by the MDR1 gene, plays an important role in the development of multidrug resistance in human hepatoblastoma (HB). Chemosensitizers antagonize the efflux action of P-glycoprotein. This study investigates the effects of 3 chemosensitizers (the cyclosporin analogue SDZ PSC 833 (PSC 833), the acridone carboxamide derivative GG 918, and verapamil) on the chemotherapy of HB in vitro. Methods: The doxorubicin (DOXO) concentration that produces 50% growth inhibition (IC50) in a HB cell line was determined and additional effects of PSC 833, GG 918, and verapamil were investigated in a cytotoxicity assay. The MDR1 gene expression after treatment was determined in a semiquantitative reverse transcription polymerase chain reaction approach. Results: The IC50 of DOXO is 2.5 μg/mL, 0.61 μg/mL for DOXO + PSC 833, 1.17 μg/mL for DOXO + verapamil, and 1.47 μg/mL for DOXO + GG 918. In combination with DOXO, cell growth was inhibited 4.1-fold by PSC 833, 2.1-fold by verapamil, and 1.9-fold by GG 918. The MDR1 gene expression was enhanced significantly in all treated cells, with and without modulator. Conclusions:MDR1 modulators significantly improve the response of HB to DOXO in vitro. The combination of anticancer agents and MDR1 modulators might be a possible contribution to overcome multidrug resistance in HB. J Pediatr Surg 37:1579-1584. Copyright 2002, Elsevier Science (USA). All rights reserved.

Application of differential display RT-PCR to identify porcine liver ESTs

Differential display banding patterns of liver and nine other tissues were produced in order to isolate porcine expressed sequence tags (ESTs), representing genes active in liver while avoiding redundant analysis of housekeeping genes. We cloned and sequenced those cDNA fragments that were unique to the liver banding pattern or that appeared in liver and a maximum of four other tissues. We analyzed 240 sequences that represent 200 distinct ESTs/genes and that make up the first list of liver ESTs in the pig. Ninety-one clones correspond to known genes and 109 clones showed no significant match with any gene or DNA sequence in GenBank and EMBL databases. Fifty-eight clones represent 18 distinct genes, the most abundant representing the albumin gene (13/240). The majority of genes that were represented by more than one clone code for proteins released by the liver into the plasma. We demonstrated the suitability of the differential display reverse transcription polymerase chain reaction approach for the detection of porcine liver ESTs. It is shown that this approach is appropriate to reduce redundant analysis of clones containing the same sequence.

Expression pattern of human P2Y receptor subtypes: a quantitative reverse transcription–polymerase chain reaction study

The diverse biological actions of extracellular nucleotides in tissues and cells are mediated by two distinct classes of P2 receptor, P2X and P2Y. The G protein-coupled P2Y receptors comprise at least six mammalian subtypes (P2Y 1,2,4,6,11,12), all of which have been cloned from human tissues, as well as other species. The P2Y receptor subtypes differ in their pharmacological selectivity for various adenosine and uridine nucleotides, which overlap in some cases. Data concerning the mRNA expression patterns of five P2Y receptors (P2Y 1,2,4,6,11) in different human tissues and cells are currently quite limited, while P2Y mRNA distribution in the human brain has not previously been studied. In this study, we have addressed this deficiency in receptor expression data by using a quantitative reverse transcription–polymerase chain reaction approach to measure the precise mRNA expression pattern of each P2Y receptor subtype in a number of human peripheral tissues and brain regions, from multiple individuals, as well as numerous human cell lines and primary cells. All five P2Y receptors exhibited widespread yet subtype-selective mRNA expression profiles throughout the human tissues, brain regions and cells used. Our extensive expression data indicate the many potentially important roles of P2Y receptors throughout the human body, and will help in elucidating the physiological function of each receptor subtype in a wide variety of human systems.

Adrenergic stimulation of osteoclastogenesis mediated by expression of osteoclast differentiation factor in MC3T3-E1 osteoblast-like cells

It is well known that adrenergic agonists efficiently activate β-adrenoceptors on osteoblastic cells and can stimulate bone resorption in intact mouse calvaria. Recently, an osteoclastogenic factor of osteoblastic origin was found to be a novel tumor necrosis factor ligand family member and was termed osteoclast differentiation factor (ODF). Using a reverse transcription–polymerase chain reaction approach, we investigated the effect of epinephrine on mRNA levels of ODF and its decoy receptor, osteoclastogenesis inhibitory factor (OCIF), in MC3T3-E1 cells. Treatment with epinephrine (1 μM) rapidly increased ODF and OCIF mRNA levels, which peaked after 0.5 hr of treatment. Epinephrine (1 μM) also increased interleukin (IL)-6, IL-11, and cyclooxygenase (COX)-II mRNA levels, as well as increased prostaglandin E 2 (PGE 2) accumulation in the culture medium. Treatment of the cells with IL-11 (10 ng/mL) or PGE 2 (1 μM) increased ODF and OCIF mRNA levels as observed with epinephrine. However, increases in ODF and OCIF mRNA levels by epinephrine were more rapid than those by IL-11, and were not influenced by NS-398 (100 μM; an inhibitor of COX-II), suggesting a direct effect of epinephrine on ODF and OCIF mRNA expressions as well as an indirect effect mediated by IL-11 and PGE 2 production. Epinephrine-induced increases in ODF and OCIF mRNA levels were inhibited by pretreatment with timolol (1 μM; β-antagonist) and phentolamine (1 μM; α-antagonist), respectively. Furthermore, the formation of tartrate-resistant acid phosphatase-positive multinucleated cells from mouse bone marrow cells was stimulated by isoproterenol (0.1 to 10 μM) or epinephrine (0.1 to 10 μM). The action of isoproterenol, a β-agonist, was clearly stronger than that of epinephrine, suggesting the importance of the physiological balance between ODF and OCIF productions for osteoclastogenesis. These findings suggest that β-adrenergic stimulation induces not only IL-6, IL-11, and PGE 2 but also ODF expression in osteoblastic cells, leading to a stimulation of osteoclastogenesis.

Cloning and immunocytochemical localization of a cyclic nucleotide-gated channel alpha-subunit to all cone photoreceptors in the mouse retina.

Cyclic nucleotide-gated channels (CNGC) are ligand-gated ion channels that open and close in response to changes in the intracellular concentration of the second messengers, 3;,5;-cyclic adenosine monophosphate and 3;,5;-cyclic guanosine monophosphate. Most notably, they transduce the chemical signal produced by the absorption of light in photoreceptors into a membrane potential change, which is then transmitted to the ascending visual pathway. CNGCs have also been implicated in the signal transduction of other neurons downstream of the photoreceptors, in particular the ON-bipolar cells, as well as in other areas of the central nervous system. We therefore undertook a search for additional cyclic nucleotide-gated channels expressed in the retina. Following a degenerate reverse transcription polymerase chain reaction approach to amplify low-copy number messages, a cDNA encoding a new splice variant of CNGC alpha-subunit was isolated from mouse retina and classified as mCNG3. An antiserum raised against the carboxy-terminal sequence identified the retinal cell type expressing mCNG3 as cone photoreceptors. Preembedding immunoelectron microscopy demonstrated its membrane localization in the outer segments, consistent with its role in phototransduction. Double-labeling experiments with cone-specific markers indicated that all cone photoreceptors in the murid retina use the same or a highly conserved cyclic nucleotide-gated channel. Therefore, defects in this channel would be predicted to severely impair photopic vision.