Microarray Method Research Articles

Graphene oxide-based NET strategy for enhanced colorimetric sensing of miRNA

MicroRNAs (miRNAs), short non-coding RNAs, have emerged as promising next-generation biomarkers of diverse diseases. In general, conventional quantitative real-time PCR and miRNA microarray methods have been utilized for the quantitative detection of miRNA. However, owing to their complicated procedures and expensive reagents/instruments, these methods cannot be widely applied by medical experts such as doctors and nurses, and are thus limited for practical clinical application. Here, we established a new graphene oxide (GO)-based NET strategy for facile miRNA detection with enhanced sensitivity, which is applicable for point-of-care testing (POCT). The proposed miRNA sensing strategy is fundamentally based on the peroxidase-mimicking DNAzyme (Dz) mediated colorimetric assay in response to target miRNA using rationally designed duplex molecular beacon (dMB). The application of GO as a net in this basic system (designated GONET) allows for gathering of the target-dependently activated Dz, which enhances the sensitivity and extends the applicability for POCT. The GONET system provides clear visualization of the target at the 10−9 M scale with the naked eye without any complicated amplification steps.

Development of a Simple and Cost-Effective Method Based on T7 Endonuclease Cleavage for Detection of Single Nucleotide Polymorphisms.

Single nucleotide polymorphisms (SNP) can be used as genetic markers and for risk assessment of allele-linked diseases, which can provide information for clinical diagnosis. Large-scale microarray and next-generation sequencing methods have made genome-wide SNP genotyping possible. However, in addition to their high cost, these techniques are dependent on having specialized equipment. Thus, there is a need for a simple genotyping method that can be implemented in a resource-limited environment. We developed a strategy for SNP genotyping based on T7 Endonuclease I cleavage and an enzyme-linked microparticle immune assay. Using this method, we genotyped two common SNP sites (rs11526468 and rs12979860). The quality of the genotyping process was validated. Although a 70% false-negative rate was observed, no false-positive reactions were found. Therefore, multiple parallel repeat reactions can offset the possibility of mutation detection failure. This method employs a duplicate reagent-dependent procedure, and therefore has the potential for integration into a portable kit for field utilization.

Rapid pathogen identification using a novel microarray-based assay with purulent meningitis in cerebrospinal fluid

In order to improve the diagnosis of pathogenic bacteria in cerebrospinal fluid (CSF) with purulent meningitis, we developed a DNA microarray technique for simultaneous detection and identification of seven target bacterium. DNA were extracted from 24 CSF samples with purulent meningitis (or suspected purulent meningitis). The specific genes of each pathogen were chosen as the amplification target, performed the polymerase chain reaction (PCR), labeled with a fluorescence dye, and hybridized to the oligonucleotide probes on the microarray. There is no significant cross-hybridization fluorescent signal occurred in untargeted bacteria. There were 87.5% (21/24) positive results in DNA microarray compared with the 58.3% (14/24) of the CSF culture test. Of which 58.3% (14/24) of the patients with culture-confirmed purulent meningitis, 37.5% (9/24) patients who were not confirmed by culture test but were demonstrated by the clinical diagnosis and DNA microarray. Multiple bacterial infections were detected in 5 cases by the microarray. In addition, the number of gene copies was carried out to determine the sensitivity of this technique, which was shown to be 3.5 × 101 copies/μL. The results revealed that the microarray technique which target pathogens of the CSF specimen is better specificity, accuracy, and sensitivity than traditional culture method. The microarray method is an effective tool for rapidly detecting more target pathogens and identifying the subtypes of strains which can eliminate the impact of the different individuals with purulent meningitis for prompt diagnosis and treatment.

Novel protein chip for the detection of antibodies against infectious bronchitis virus

BackgroundInfectious bronchitis (IB) caused by the IB virus (IBV) can cause acute damage to chickens around the world. Therefore, rapid diagnosis and immune status determination are critical for controlling IBV outbreaks. Enzyme-linked immunosorbent assays (ELISAs) have been widely used in the detection of IBV antibodies in the early infection and continuous infection of IB because they are more sensitive and quicker than other diagnostic methods.ResultsWe have developed two indirect microarray methods to detect antibodies against IBV: a chemiluminescent immunoassay test (CIT) and a rapid diagnostic test (RDT). IBV nonstructural protein 5 (nsp5) was expressed, purified from Escherichia coli, and used to spot the initiator integrated poly(dimethylsiloxane), which can provide a near “zero” background for serological assays. Compared with the IDEXX IBV Ab Test kit, CIT and RDT have a sensitivity and specificity of at least 98.88% and 91.67%, respectively. No cross-reaction was detected with antibodies against avian influenza virus subtypes (H5, H7, and H9), Newcastle disease virus, Marek’s disease virus, infectious bursal disease virus, and chicken anemia virus. The coefficients of variation of the reproducibility of the intra- and inter-assays for CIT ranged from 0.8 to 18.63%. The reproducibility of RDT was consistent with the original results. The application of the IBV nsp5 protein microarray showed that the positive rate of the CIT was 96.77%, that of the nsp5 ELISA was 91.40%, and that of the RDT was 90.32%. Furthermore, the RDT, which was visible to the naked eye, could be completed within 15 min. Our results indicated that compared with nsp5 ELISA, the CIT was more sensitive, and the RDT had similar positive rates but was faster. Furthermore, the two proposed methods were specific and stable.ConclusionsTwo microarray assays, which were rapid, specific, sensitive, and relatively simple, were developed for the detection of an antibody against IBV. These methods can be of great value for the surveillance of pathogens and monitoring the efficiency of vaccination.

Exploring the molecular mechanism associated with breast cancer bone metastasis using bioinformatic analysis and microarray genetic interaction network.

Background:Bone metastases are common in advanced breast cancer patients and frequently leading to skeletal-related morbidity and deterioration in the quality of life. Although chemotherapy and hormone therapy are able to control the symptoms caused by bone destruction, the underlying molecular mechanisms for the affinity of breast cancer cells towards skeletal bones are still not completely understood.Methods:In this study, bioinformatic analysis was performed on patients’ microarray gene expression data to explore the molecular mechanism associated with breast cancer bone metastasis. Microarray gene expression profile regarding patients with breast cancer and disseminated tumor cells was downloaded from Gene Expression Omnibus (GEO) database (NCBI, NIH). Raw data were normalized and differently expressed genes were identified by using Significance Analysis of Microarrays (SAM) methods. Protein interaction networks were expanded using String. Moreover, molecular functions, biological processes and signaling pathway enrichment analysis were performed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG).Results:We identified 66 differentially expressed genes. After submitting the set of genes to String, genetic interaction network was expanded, which consisted of 110 nodes and 869 edges. Pathway enrichment analysis suggested that adhesion kinase, ECM-receptor interaction, calcium signaling, Wnt pathways, and PI3K/AKT signaling pathway are highly associated with breast cancer bone metastasis.Conclusion:In this study, we established a microarray genetic interaction network associated with breast cancer bone metastasis. This information provides some potential molecular therapeutic targets for breast cancer initiation and progression.

OR34 Modernizing ABO antibody detection: Development of a bead-based ABO antibody detection assay

Aim Accurate, clinically relevant characterization of ABO antibodies (ABO-Ab) is critical to assess their impact in ABO incompatible transplantation. The current ABO-Ab detection method using erythrocyte agglutination is limited by lack of ABO subtype specificity, difficulty in ABO-Ab isotype differentiation, and poor reproducibility. We previously developed a slide micro-array method for ABO-Ab analysis to address these limitations. Our aim was to create a similar bead solid phase assay. Methods ABO A subtype antigens (I, II, III, IV, V, VI) were coupled to Luminex beads and quantified using monoclonal ABO-Ab. Bovine serum albumin and alpha-Gal antigen were coupled as negative/positive control beads, respectively. Optimal plasma and anti-human IgG and IgM labelling-antibody dilutions were determined. IgG and IgM isotypes with specificities for ABO A-subtypes were measured (n = 40 healthy donors) by mean fluorescent intensity (MFI). Levels of IgG vsIgM ABO-A-Ab were compared (Wilcoxon signed-rank test). Preliminary positive MFI thresholds for each ABO A-subtype bead were calculated based on ABO-A-Ab in ABO A control plasma. Results Variation in ABO-Ab levels between ABO A-subtypes was detected. There was no significant difference between IgG and IgM ABO-Ab levels in paired MFI for all ABO types. IgG and IgM ABO-Ab are clearly detectable in all non-ABO A controls. Calculated MFI thresholds accurately interpreted positive ABO-A-Ab in ABO O samples for the IgG assay and most beads in the IgM assay. ABO B controls had similar IgM ABO-A-Ab levels to ABO O controls but lower levels of IgG. Reactivity to alpha-Gal was present but inconsistent. Conclusions This bead-based method successfully measures ABO-A-Ab and shows promise for clinical laboratory implementation. The specificity of these solid-phase assays will facilitate accurate assessment of ABO-Ab to subtypes, which are known to be expressed differently in endothelium than on erythrocytes. The ability to accurately measure both IgM and IgG ABO antibodies makes it possible to evaluate of the role of each ABO-Ab isotype in transplantation; this may be particularly relevant in the setting of plasmapheresis, which more efficiently removes IgM antibodies. Further optimization of this assay, controls, and ABO B and ABO H panel development are underway.

Analysis of serum alpha-fetoprotein (AFP) and AFP-L3 levels by protein microarray.

ObjectivesThis study aimed to examine a simple, effective, time-saving, and low-cost protein microarray method for detecting serum alpha-fetoprotein (AFP) and AFP-L3 levels.MethodsSerum samples from patients with hepatocellular carcinoma (HCC) (n = 33) and control subjects (n = 39) were collected and evaluated for the presence of AFP using a novel protein microarray. Glycoprotein (including AFP-L3) was enriched from crude samples by a Hotgen Biotech glycosyl capture spin column and then detected by protein microarray. An electrochemiluminescence immunoassay (ECLIA) was used to validate the measured values.ResultsNeither AFP levels lower than 20 ng/mL in the HCC group nor AFP levels higher than 20 ng/mL in the control group were found when tested by the ECLIA and protein microarray. The kappa test showed good consistency in the diagnostic performance of measuring serum AFP levels and the percentage of AFP-L3 in total AFP by the ECLIA and protein microarray. Protein microarray had advantages of smaller sample size required, low cost, and convenience compared with the ECLIA.ConclusionThe protein microarray assay that was developed in the present study shows potential as an economic and convenient technique for detecting AFP and AFP-L3 levels in serum samples from patients with HCC.

Development of the microarray tecnology for investigation of antibody responses during Zika virus infection and of anti-Flaviviruses IgM/IgG cross-reactivity

Background: Short viremia period of Zika virus leads to the necessity of the indirect serological diagnostics. To overcome Flavivirus cross-reactivity, recombinant envelope (E) and NS1 proteins are commonly used. Here we aimed to investigate the terms of IgM and IgG circulation in serum samples and it‘s cross-reactivity between Flaviviruses (TBEV, DV, WNFV). Methods & Materials: The study was conducted using sera from 9 patients (total 56 samples) in which ZIKV RNA was determined using the «AmpliSens® Zika virus-FL» system (CRIE, Russia). Immunoglobulin levels were measured using developed microarray method. Results: Only IgM to NS1 ZIKV protein were discovered in the first days (3-5 day) after the onset of disease symptoms (fever 9/9, joint pain 9/9, rash 5/9, conjunctivitis 2/9). IgG to one or both ZIKV proteins were discovered on 5-8 day in all 9 cases (3/9 anti-E protein, 5/9 anti-NS1 protein, in 1/9 case to both ZIKV antigens). 7/9 subjects were followed-up longer than 3 month. In 3/7 cases IgM to ZIKV NS1 have been discovered during more than 3 months, in 4/7 cases only during 8-21 days. IgG to ZIKV proteins have been discovered in serum samples during more than 12 months. IgM/IgG to ZIKV antigens weren’t found in reference cohort (50 samples) under assay conditions. Cross-reactivity of E protein was detected in 4/14, 12/12, 15/35 cases IgG positive to TBEV, DV and WNFV respectively and of NS1 protein only in 3/12 and 2/35 samples IgG positive to DV and WNFV respectively. No TBEV, 1/33 of DV and 3/48 of WNFV IgM positive samples cross-reacted with ZIKV proteins, and only 1/33 of DV and 1/48 of WNFV IgM positive sample contained anti-NS1 IgM. Conclusion: We showed that developed microarray containing ZIKV E and NS1 proteins is highly sensitive and specific diagnostic mean. When IgM response to ZIKV should be investigated the usage of NS1 protein alone provides high sensitivity and prevents cross-reactions with antibodies to other flaviviruses. For highest IgG sensitivity both NS1 and E ZIKV proteins should be used. But exclusively IgG response does not allow infection determination due to cross-reactivity between flaviviruses. This study was supported by the Russian Scientific Fund grant #17-75-10093.

Potential Mechanism of Action of Cyclosporin A in Human Dermal Fibroblasts-Transcriptomic Analysis of CYPs.

Effect of cyclosporin A (CsA) in a therapeutic concentration, on the expression of cytochrome P450 genes (CYPs), was investigated in normal human dermal fibroblast cells. The expression of 57 genes, encoding cytochrome P450 isoforms, was estimated using the microarray method. Amongst 396 normalized fluorescence signals related to cytochrome P450 activity, only 91 were strictly connected to CYPs and were analyzed using two methods: a self-organizing feature map of artificial neural networks and typical statistical analysis with significance level at p ≤ 0.05. Comparing the samples from fibroblasts cultured with CsA and those cultured without, up-regulated changes of CYP19A1, 1B1, 7A1, 7F1, 17A1 and down-regulated 2D6 gene expression were observed. The mRNAs with increased changes were in the same neuron of the self-organizing feature map. All distinguished CYPs encode monooxygenases, which plays an important role in steroids biosynthesis and metabolism. Based on the obtained results, we can conclude that CsA in therapeutic concentration changes the expression profile of CYPs in human dermal fibroblasts, especially affecting genes linked to steroids synthesis and/or metabolism. It shows the potential mechanism of action of CsA in human dermal fibroblast cells.

Changes in Differential Expression of Genes in Normal and Metabolically Suppressed Mice in Response to Radiation.

Differential gene expression profiles of mice in active and metabolically suppressed states were evaluated in response to a sublethal dose of gamma radiation to identify the beneficial protective effects of suspended animation. Results demonstrated that nearly 90% suppression of metabolic functions in mice lead to significant changes in gene expression profile of different metabolic pathways responsible for adaptation and maintenance of homeostasis in the new physiological state of suspended animation. This state was found sustainable during 18 hrs of experiment and can be reversed back to the normal active state without any visible effects on physiological and behavioral functions of mice. Gene expression during induced states was gathered via Illumina microarray methods. Further analysis of differential gene expressions yielded a result that a hypometabolic state may be responsible for short term and long-term radioprotection.

Human-associated microbial populations as evidence in forensic casework

In forensic investigations involving human biological traces, cell type identification is often required. Identifying the cell type from which a human STR profile has originated can assist in verifying scenarios. Several techniques have been developed for this purpose, most of which focus on molecular characteristics of human cells. Here we present a microarray method focusing on the microbial populations that are associated with human cell material. A microarray with 863 probes targeting (sets of) species, specific genera, groups of genera or families was designed for this study and evaluated with samples from different body sites: hand, foot, groin, penis, vagina, mouth and faeces. In total 175 samples from healthy individuals were analysed. Next to human faeces, 15 feline and 15 canine faeces samples were also included. Both clustering and classification analysis were used for data analysis. Faecal and oral samples could clearly be distinguished from vaginal and skin samples, and also canine and feline faeces could be differentiated from human faeces. Some penis samples showed high similarity to vaginal samples, others to skin samples. Discriminating between skin samples from different skin sites proved to be challenging. As a proof of principle, twenty-one mock case samples were analysed with the microarray method. All mock case samples were clustered or classified within the correct main cluster/group. Only two of the mock case samples were assigned to the wrong sub-cluster/class; with classification one additional sample was classified within the wrong sub-class. Overall, the microarray method is a valuable addition to already existing cell typing techniques. Combining the results of microbial population analysis with for instance mRNA typing can increase the evidential value of a trace, since both techniques focus on independent targets within a sample.

Influence of Glucose Availability and CRP Acetylation on the Genome-Wide Transcriptional Response of Escherichia coli: Assessment by an Optimized Factorial Microarray Analysis.

Background: While in eukaryotes acetylation/deacetylation regulation exerts multiple pleiotropic effects, in Escherichia coli it seems to be more limited and less known. Hence, we aimed to progress in the characterization of this regulation by dealing with three convergent aspects: the effector enzymes involved, the master regulator CRP, and the dependence on glucose availability.Methods: The transcriptional response of E. coli BW25113 was analyzed across 14 relevant scenarios. These conditions arise when the wild type and four isogenic mutants (defective in deacetylase CobB, defective in N(ε)-lysine acetyl transferase PatZ, Q- and R-type mutants of protein CRP) are studied under three levels of glucose availability (glucose-limited chemostat and glucose-excess or glucose-exhausted in batch culture). The Q-type emulates a permanent stage of CRPacetylated, whereas the R-type emulates a permanent stage of CRPdeacetylated. The data were analyzed by an optimized factorial microarray method (Q-GDEMAR).Results: (a) By analyzing one mutant against the other, we were able to unravel the true genes that participate in the interaction between ΔcobB/ΔpatZ mutations and glucose availability; (b) Increasing stages of glucose limitation appear to be associated with the up-regulation of specific sets of target genes rather than with the loss of genes present when glucose is in excess; (c) Both CRPdeacetylated and CRPacetylated produce extensive changes in specific subsets of genes, but their number and identity depend on the glucose availability; (d) In other sub-sets of genes, the transcriptional effect of CRP seems to be independent of its acetylation or deacetylation; (e) Some specific ontology functions can be associated with each of the different sets of genes detected herein.Conclusions: CRP cannot be thought of only as an effector of catabolite repression, because it acts along all the glucose conditions tested (excess, limited, and exhausted), exerting both positive and negative effects through different sets of genes. Acetylation of CRP does not seem to be a binary form of regulation, as there is not a univocal relationship between its activation/inhibitory effect and its acetylation/deacetylation stage. All the combinatorial possibilities are observed. During the exponential growth phase, CRP also exerts a very significant transcriptional effect, mainly on flagellar assembly and chemotaxis (FDR = 7.2 × 10−44).

Circular RNA expression profiles in hippocampus from mice with perinatal glyphosate exposure

Glyphosate is the active ingredient in numerous herbicide formulations. The roles of glyphosate in embryo-toxicity and neurotoxicity have been reported in human and animal models. Recently, several studies have reported evidence linking neurodevelopmental disorders (NDDs) with gestational glyphosate exposure. However, the role of glyphosate in neuronal development is still not fully understood. Our previous study found that perinatal glyphosate exposure resulted in differential microRNA expression in the prefrontal cortex of mouse offspring. However, the mechanism of glyphosate-induced neurotoxicity in the developing brain is still not fully understood. Considering the pivotal role of Circular RNAs (circRNAs) in the regulation of gene expression, a circRNA microarray method was used in this study to investigate circRNA expression changes in the hippocampus of mice with perinatal glyphosate exposure. The circRNA microarrays revealed that 663 circRNAs were significantly altered in the perinatal glyphosate exposure group compared with the control group. Among them, 330 were significantly upregulated, and the other 333 were downregulated. Furthermore, the relative expression levels of mmu-circRNA-014015, mmu-circRNA-28128 and mmu-circRNA-29837 were verified using quantitative real-time polymerase chain reaction (qRT-PCR). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses demonstrated that stress-associated steroid metabolism pathways, such as aldosterone synthesis and secretion pathways, may be involved in the neurotoxicity of glyphosate. These results showed that circRNAs are aberrantly expressed in the hippocampus of mice with perinatal glyphosate exposure and play potential roles in glyphosate-induced neurotoxicity.

Integrating Exosomal MicroRNA and Electronic Health Data Improves Tuberculosis Diagnosis

Background: Tuberculosis is difficult to diagnose under complex clinical conditions. Diagnostic information from electronic health records (EHRs) remains insufficient. Exosomal miRNAs have emerged as promising disease biomarkers. We aimed to investigate the potential of exosomal miRNAs and EHRs to assist with the clinical diagnosis of tuberculosis. Methods: We recruited 407 individuals through a multi-stage approach. Exosomal miRNA expression was profiled using microarray and qRT-PCR method. Differentially expressed miRNAs (DEMs) were selected in the discovery set and validated in the prospective selection and independent testing cohorts. EHRs and follow-up information were collected accordingly. The 'EHR miRNA', 'miRNA only' and 'EHR only' models were established using the Support Vector Machine algorithm. Findings: A total of 370 individuals were ultimately enrolled. Six DEMs (miR-20a, miR-20b, miR-26a, miR-106a, miR-191, and miR-486) were found to be overexpressed in pulmonary tuberculosis (PTB) and tuberculous meningitis (TBM) patients relative to their controls. The 'EHR miRNA' model showed a superior clinical diagnostic efficacy, with an increased AUC of 0.97 (95% CI 0.80-0.99) in TBM and 0.97 (0.87-0.99) in PTB, respectively. Model feature analysis determined that miR-20b, miR-191, and miR-486 were promising diagnostic biomarkers for tuberculosis. The expression of DEMs presented a decreased trend after 2-month intensive anti-tuberculosis therapy (adjusted p = 4.80 ×10-5). Interpretation: Our results concluded that the combination of exosomal miRNAs and EHRs in a machine learning algorithm potentially improved TBM and PTB clinical diagnoses. Further validation is warranted. Funding Statement: Funds for the Central Universities, the National Natural Science Foundation of China. Declaration of Interests: The authors declare that they have no competing interests. Ethics Approval Statement: The study was approved by the Clinical Trials and Biomedical Ethics Committee of West China Hospital, Sichuan University [No. 2014 (198)]. Informed consent was obtained from the participants.

Identification of age-related changes in gene expression and chromatin accessibility in T cells from thymus to periphery

Abstract Aging of immune system is characterized by progressive decline of physiological and cellular function of T cells. Recent studies of naïve T cells from young and old mice have identified age-related altered gene expressions but the mechanisms underlying age associated changes of naïve T cells remain poorly understood. Here we compared the transcriptome and chromatin accessibility in mature CD4 and CD8 thymocytes and in naïve CD4 and CD8 T cells of spleen between young (6–8 weeks) and old (95–110 weeks) C57Bl/6 mice. We used Agilent microarray method and identified the age-related altering gene expressions 1) only in mature CD4 and CD8 T cell from thymus, 2) shared by both thymus and spleen naïve T cells, and 3) only in naïve T cells from spleen. The shared changes include increased chemokine and chemokine receptor expressions and decreased cell cycle regulator expression. We further analyzed the chromatin basis of altered gene expression using ATAC seq between young and old mice. We have identified age-related changes of chromatin accessibility in T cells from thymus and spleen, and some correlated changes between gene expression and chromatin accessibility. Overall, we observed more age-related alterations of gene expression and chromatin accessibility in thymus than in spleen, suggesting some age-related changes of T cells in thymus did not retain in periphery. Together, our findings have identified tissue specific altered gene expressions and chromatin status in T cells during aging and further study of these altered genes will shed new insights into the mechanisms underlying these age-related changes. This research was supported in part by the Intramural Research Program of the NIH, National Institute on Aging.

Fabrication of agarose concave petridish for 3D-culture microarray method for spheroids formation of hepatic cells.

Liver is one of the most important organ in the body. But there are many limitations about liver transplantation for liver failure. It is quite important to develop the xenogeneic biological liver for providing an alternation to transplantation or liver regeneration. In this paper, we proposed a method to construct a novel kind of agarose 3D-culture concave microwell array for spheroids formation of hepatic cells. Using the 3D printing method, the microwell array was fabricated with an overall size of 6.4 mm × 6.4 mm, containing 121 microwells with 400 μm width/400 μm thickness. By exploiting the Polydimethylsiloxane (PDMS) membranes as a bridge, we finally fabricated the agarose one. We co-cultured three types of liver cells with bionics design in the microwell arrays. Using the methods described above, the resulting co-formed hepatocyte spheroids maintained the high viability and stable liver-specific functions. This engineered agarose concave microwell array could be a potentially useful tool for forming the elements for biological liver support. After developing the complete system, we also would consider to scale up the application of this system. It will be not only applied to the therapy of human organ damage, but also to the development of disease models and drug screening models.

TiO2 Nanolayer-Enhanced Fluorescence for Simultaneous Multiplex Mycotoxin Detection by Aptamer Microarrays on a Porous Silicon Surface.

A new aptamer microarray method on the TiO2-porous silicon (PSi) surface was developed to simultaneously screen multiplex mycotoxins. The TiO2 nanolayer on the surface of PSi can enhance the fluorescence intensity 14 times than that of the thermally oxidized PSi. The aptamer fluorescence signal recovery principle was performed on the TiO2-PSi surface by hybridization duplex strand DNA from the mycotoxin aptamer and antiaptamer, respectively, labeled with fluorescence dye and quencher. The aptamer microarray can simultaneously screen for multiplex mycotoxins with a dynamic linear detection range of 0.1-10 ng/mL for ochratoxin A (OTA), 0.01-10 ng/mL for aflatoxins B1 (AFB1), and 0.001-10 ng/mL for fumonisin B1 (FB1) and limits of detection of 15.4, 1.48, and 0.21 pg/mL for OTA, AFB1, and FB1, respectively. The newly developed method shows good specificity and recovery rates. This method can provide a simple, sensitive, and cost-efficient platform for simultaneous screening of multiplex mycotoxins and can be easily expanded to the other aptamer-based protocol.

Gene expression profiling of rat livers after continuous whole-body exposure to low-dose rate of gamma rays

Purpose: To study gene expression modulation in response to continuous whole-body exposure to low-dose-rate gamma radiation and improve our understanding of the mechanism of this impact at the molecular basis.Materials and methods: cDNA microarray method with complete pooling of samples was used to study expression changes in the transcriptome profile of livers from rats treated with prolonged low-dose-rate ionizing radiation (IR) relative to that of sham-irradiated rats.Results: Of the 209 genes that were two-fold-up or down-regulated, 143 were known genes of which 27 were found in previous literatures to be modulated by IR. Remarkably, there were a significant number of differentially expressed genes involved in hepatic lipid metabolism.Conclusion: This study showed changes in transcriptome profile of livers from low-dose irradiated rats when compared with that of sham-irradiated ones. This study will be useful for studying the metabolic changes of human exposed for long term to cosmic ray such as in space and in polar regions.

Comparison of the Diagnostic Accuracy of Next Generation Sequencing and Microarray Resequencing Methods for Detection of BRCA1 and BRCA2 Gene Mutations

Objective: Breast cancer constitutes 29 % of estimated new cases of cancer in women, and it is also one of the major cause of death in all cancer types. In this study, DNA samples of familial breast cancer patients with BRCA1 and BRCA2 mutations which had been analyzed using conventional DNA sequencing method, were also analyzed with new methods including microarray and next generation sequencing (NGS) in order to compare their results Methods: Seven patients with BRCA1 mutation, one patient with BRCA2 mutation, and two controls were included. All samples for the microarray method were studied on the GeneChip 3000 Scanner (Affymetrix) system and then analyzed on the Affymetrix GeneChip Resequencing Analysis Software (GSEQ® v4.0) system. Four patients from the patient group were selected for next generation sequencing and were analyzed on GS Junior 454 (Roche, Prague, Czech Republic) system. The raw data had been analysed by SeqPilot SeqNext module (v4.0, JSI medical systems, Kippenheim, Germany). Results: Microarray resequencing analysis did not detect the mutations defined by conventional sequencing in patients, but mutations were detected in all of the 4 patients in the next generation sequencing. Conclusion: Our study detected the NGS to be reliable as conventional DNA sequencing for studying BRCA1/BRCA2 gene mutations. However, we suggest to confirm the NGS results with a conventional method because of homopolymer sequences which may cause false positive results.

Identification of novel loci associated with maturity and yield traits in early maturity soybean plant introduction lines

BackgroundTo continue to meet the increasing demands of soybean worldwide, it is crucial to identify key genes regulating flowering and maturity to expand the cultivated regions into short season areas. Although four soybean genes have been successfully utilized in early maturity breeding programs, new genes governing maturity are continuously being identified suggesting that there remains as yet undiscovered loci governing agronomic traits of interest. The objective of this study was to identify novel loci and genes involved in a diverse set of early soybean maturity using genome-wide association (GWA) analyses to identify loci governing days to maturity (DTM), flowering (DTF) and pod filling (DTPF), as well as yield and 100 seed weight in Canadian environments. To do so, soybean plant introduction lines varying significantly for maturity, but classified as early varieties, were used. Plants were phenotyped for the five agronomic traits for five site-years and GWA approaches used to identify candidate loci and genes affecting each trait.ResultsGenotyping using genotyping-by-sequencing and microarray methods identified 67,594 single nucleotide polymorphisms, of which 31,283 had a linkage disequilibrium < 1 and minor allele frequency > 0.05 and were used for GWA analyses. A total of 9, 6, 4, 5 and 2 loci were detected for GWA analyses for DTM, DTF, DTPF, 100 seed weight and yield, respectively. Regions of interest, including a region surrounding the E1 gene for flowering and maturity, and several novel loci, were identified, with several loci having pleiotropic effects. Novel loci affecting maturity were identified on chromosomes five and 13 and reduced maturity by 7.2 and 3.3 days, respectively. Novel loci for maturity and flowering contained genes orthologous to known Arabidopsis flowering genes, while loci affecting yield and 100 seed weight contained genes known to cause dwarfism.ConclusionsThis study demonstrated substantial variation in soybean agronomic traits of interest, including maturity and flowering dates as well as yield, and the utility of GWA analyses in identifying novel genetic factors underlying important agronomic traits. The loci and candidate genes identified serve as promising targets for future studies examining the mechanisms underlying the related soybean traits.