Development and use of a droplet digital PCR (ddPCR) assay to achieve sensitive and fast atypical porcine pestivirus detection.

Atypical porcine pestivirus (APPV) is a recently discovered RNA virus, which mainly caused congenital tremor in piglets. Droplet digital PCR (ddPCR) is an absolute quantitative method that does not rely on the standard curve but has high sensitivity and accuracy. The present study aimed to develop a ddPCR detection assay for APPV. Furthermore, we evaluated the limit of detection, sensitivity, specificity and reproducibility of the ddPCR and real-time quantitative PCR (qPCR) and tested 135 clinical samples to calculate the detection rate of the two methods. The results showed that both methods had a strong linear relationship and quantitative correlation. The ddPCR assay had a minimum detection limit of 0.15 copies/µL for APPV, with a sensitivity 100 times that of qPCR. We tested clinical samples and found that the APPV ddPCR had a 27.4% positive detection rate, noticeably higher than that of the qPCR (14.8%). Additionally, the APPV ddPCR method had excellent repeatability and specificity. In brief, our study provided a novel, feasible and sensitive diagnostic technique to identify and monitor APPV.

Validation and Selection of New Reference Genes for RT-qPCR Analysis in Pediatric Glioma of Different Grades.

Gliomas are heterogeneous, solid, and intracranial tumors that originate from glial cells. Malignant cells from the tumor undergo metabolic alterations to obtain the energy required for proliferation and the invasion of the cerebral parenchyma. The alterations in the expression of the genes related to the metabolic pathways can be detected in biopsies of gliomas of different CNS WHO grades. In this study, we evaluated the expression of 16 candidate reference genes in the HMC3 microglia cell line. Then, statistical algorithms such as BestKeeper, the comparative ΔCT method, geNorm, NormFinder, and RefFinder were applied to obtain the genes most suitable to be considered as references for measuring the levels of expression in glioma samples. The results show that PKM and TPI1 are two novel genes suitable for genic expression studies on gliomas. Finally, we analyzed the expression of genes involved in metabolic pathways in clinical samples of brain gliomas of different CNS WHO grades. RT-qPCR analysis showed that in CNS WHO grade 3 and 4 gliomas, the expression levels of HK1, PFKM, GAPDH, G6PD, PGD1, IDH1, FASN, ACACA, and ELOVL2 were higher than those of CNS WHO grade 1 and 2 glioma biopsies. Hence, our results suggest that reference genes from metabolic pathways have different expression profiles depending on the stratification of gliomas and constitute a potential model for studying the development of this type of tumor and the search for molecular targets to treat gliomas.

Octahedral ruthenium and magnesium naringenin 5-alkoxide complexes: NMR analysis of diastereoisomers and in-vivo antibacterial activity against Xylella fastidiosa.

Although many copper-based antimicrobial compounds have been developed to control pathogenic bacteria and fungi in plants and applied for crop protection, there is evidence that several plant pathogens have developed resistance to copper-based antimicrobial compounds, including some Xanthomonas species. Xylella is a bacterial genus belonging to the Xanthomonas family; and X. fastidiosa, which is responsible for citrus variegated chlorosis (CVC) in sweet orange, may develop resistance to one or more copper-based antimicrobials. Because of the time required for the development and approval of new antimicrobials for commercial use, the discovery of novel bactericidal compounds is essential before the development of resistance to the antimicrobials currently in use becomes widespread. Here, we explored the antimicrobial potential of two newly synthesized antimicrobials complexes and one natural compound against X. fastidiosa. Several nuclear magnetic resonance (NMR) assays with high resolution and sensitivity were developed to identify new diastereoisomers in the context of octahedral ruthenium - [Ru(narin)(phen)2]PF6-and magnesium naringenin 5-alkoxide - [Mg(narin)(phen)2]OAc - complexes, obtained in the present work. The NMR assays proved to be powerful tools for the identification of isomers in metal complexes. Moreover, a protocol for the in-vivo determination of the effects of these complexes against X. fastidiosa was developed. The main trunks of X. fastidiosa infected plants were injected with the two complexes as well as with the limonoid azadirachtin using a syringe; the number of bacterial cells in the plants following treatment was estimated via real-time quantitative PCR (qPCR). Importantly, the administration of both complexes and of azadirachtin drastically reduced the number of X. fastidiosa cells in vivo.

Modulation of Cytokines and Extracellular Matrix Proteins Expression by Leishmania amazonensis in Susceptible and Resistant Mice.

Leishmaniases are a complex of diseases with a broad spectrum of clinical forms, which depend on the parasite species, immunological status, and genetic background of the host. In the Leishmania major model, susceptibility is associated with the Th2 pattern of cytokines production, while resistance is associated with Th1 response. However, the same dichotomy does not occur in L. amazonensis-infected mice. Cytokines are key players in these diseases progression, while the extracellular matrix (ECM) components participate in the process of parasite invasion as well as lesion healing. In this article, we analyzed the influence of host genetics on the expression of cytokines, inducible nitric oxide synthase (iNOS), and ECM proteins, as well as the parasite load in mice with different genetic backgrounds infected by L. amazonensis. C57BL/10 and C3H/He mice were subcutaneously infected with 106 L. amazonensis promastigotes. Lesion kinetics, parasite load, cytokines, iNOS, and ECM proteins expression were measured by quantitative PCR (qPCR) in the footpad, draining lymph nodes, liver, and spleen at early (24 h and 30 days) and late phase (120 and 180 days) of infection. Analysis of lesion kinetics showed that C57BL/10 mice developed ulcerative lesions at the inoculation site after L. amazonensis infection, while C3H/He showed slight swelling in the footpad 180 days after infection. C57BL/10 showed progressive enhancement of parasite load in all analyzed organs, while C3H/He mice showed extremely low parasite loads. Susceptible C57BL/10 mice showed high levels of TGF-ß mRNA in the footpad early in infection and high levels of proinflammatory cytokines mRNA (IL-12, TNF-α, and IFN-γ) and iNOS in the late phase of the infection. There is an association between increased expression of fibronectin, laminin, collagen III and IV, and TGF-ß. On the other hand, resistant C3H/He mice presented a lower repertory of cytokines mRNA expression when compared with susceptible C57BL/10 mice, basically producing TNF-α, collagen IV, and laminin early in infection. The findings of our study indicate that L. amazonensis infection induces different cytokine expression in resistant and susceptible mice but not like the L. major model. An organ-compartmentalized cytokine response was observed in our model. Host genetics determine this response, which modulates ECM proteins expression.

Quantitative real-time PCR for differential diagnostics of parvovirus B19 infection in acute liver failure patients.

BACKGROUND: Human Parvovirus B19 (B19V) is a common pathogen worldwide. After primary infection, B19V-DNA may permanently persist in non-erythroid tissues, including the liver of patients with acute liver failure (ALF). OBJECTIVE: To validate a real-time PCR (qPCR) for the quantification of B19V-DNA, in order to establish a differential diagnosis for B19V infection in ALF patients. METHODS: The qPCR techniques were based on Sybr Green® and TaqMan® methodologies. To evaluate the quality parameters of both methods, samples from patients with or without B19V infection were tested. The diagnostic utility of qPCR in the detection B19V-DNA in patients with ALF was evaluated by testing archived serum and hepatic tissue explants from 10 patients. RESULTS: The Sybr Green® methodology showed 97% efficiency, the limits of detection and quantification were 62.6 and 53,200 copies/mL, respectively. The TaqMan® methodology showed 95% efficiency, the limits of detection and quantification were 4.48 and 310 copies/mL, respectively. A false positive result was found only with the Sybr Green® methodology. Among ALF patients without defined etiology, three (30%) were positive for B19V DNA in serum and liver. CONCLUSION: The qPCR methods validated here were effective in clarifying uncommon cases of B19V-related ALF and are fit for differential diagnosis of ALF causes.

Evaluation of the transcriptional immune biomarkers in peripheral blood from warao indigenous associate with the infection by mycobacterium tuberculosis

Abstract INTRODUCTION: Biomarkers are critical tools for finding new approaches for controlling the spread of tuberculosis (TB), including for predicting the development of TB therapeutics, vaccines, and diagnostic tools. METHODS: Expression of immune biomarkers was analyzed in peripheral blood cells stimulated and non-stimulated with M. tuberculosis antigens ESAT-6, CFP10 and TB7.7. in Warao indigenous individuals. These biomarkers may be able to differentiate TB states, such as active tuberculosis (ATB) cases and latent tuberculosis infection (LTBI) from non-infected controls (NIC). A real-time reverse transcription polymerase chain reaction (RT-qPCR) assay was performed on 100 blood samples under non-stimulation or direct ex vivo conditions (NS=50) and stimulation conditions (S=50). RESULTS: The findings are shown as the median and interquartile range (IQR) of relative gene expression levels of IFN-γ, CD14, MMP9, CCR5, CCL11, CXCL9/MIG, and uPAR/PLAUR immune biomarkers. MMP9 levels were significantly higher in the LTBI-NS and LTBI-S groups compared with the NIC-NS and NIC-S groups. However, CCR5 levels were significantly lower in the LTBI-S group compared with both NIC-NS and NIC-S groups. CCL11 levels were significantly lower in the LTBI-S group compared with the NIC-NS group. CONCLUSIONS: Preliminary findings showed that MMP9 immune biomarkers separated LTBI indigenous individuals from NIC indigenous individuals, while CCR5, CCL11, CD14, and IFN-γ did not differentiate TB states from NIC. MMP9 may be useful as a potential biomarker for LTBI and new infected case detection among Warao indigenous individuals at high risk of developing the disease. It may also be used to halt the epidemic, which will require further validation in larger studies.

Strain-dependent mutational effects for Pepino mosaic virus in a natural host.

BACKGROUND: Pepino mosaic virus (PepMV) is an emerging plant pathogen that infects tomatoes worldwide. Understanding the factors that influence its evolutionary success is essential for developing new control strategies that may be more robust against the evolution of new viral strains. One of these evolutionary factors is the distribution of mutational fitness effect (DMFE), that is, the fraction of mutations that are lethal, deleterious, neutral, and beneficial on a given viral strain and host species. The goal of this study was to characterize the DMFE of introduced nonsynonymous mutations on a mild isolate of PepMV from the Chilean 2 strain (PepMV-P22). Additionally, we also explored whether the fitness effect of a given mutation depends on the gene where it appears or on epistatic interactions with the genetic background. To address this latter possibility, a subset of mutations were also introduced in a mild isolate of the European strain (PepMV-P11) and the fitness of the resulting clones measured. RESULTS: A collection of 25 PepMV clones each containing a single nucleotide nonsynonymous substitution was created by site-directed mutagenesis and the fitness of each mutant was determined. PepMV-P22 genome showed a high degree of robustness against point mutations, with 80% of mutations being either neutral or even beneficial and only 20% being deleterious or lethal. We found that the effect of mutations strongly depended on the gene in which they were introduced. Mutations with the largest average beneficial effects were those affecting the RdRp gene, in contrast to mutations affecting TGB1 and CP genes, for which the average effects were deleterious. Moreover, significant epistatic interactions were observed between nonsynonymous mutations and the genetic background, meaning that the effect of a given nucleotide substitution on a particular genomic context cannot be predicted by knowing its effect in a different one. CONCLUSIONS: Our results indicated that PepMV genome has a surprisingly high robustness against mutations. We also found that fitness consequences of a given mutation differ between the two strains analyzed. This discovery suggests that the strength of selection, and thus the rates of evolution, vary among PepMV strains.

Evaluation of stability and validation of reference genes for RT-qPCR expression studies in rice plants under water deficit.

Many studies use strategies that allow for the identification of a large number of genes expressed in response to different stress conditions to which the plant is subjected throughout its cycle. In order to obtain accurate and reliable results in gene expression studies, it is necessary to use reference genes, which must have uniform expression in the majority of cells in the organism studied. RNA isolation of leaves and expression analysis in real-time quantitative polymerase chain reaction (RT-qPCR) were carried out. In this study, nine candidate reference genes were tested, actin 11 (ACT11), ubiquitin conjugated to E2 enzyme (UBC-E2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta tubulin (ß-tubulin), eukaryotic initiation factor 4α (eIF-4α), ubiquitin 10 (UBQ10), ubiquitin 5 (UBQ5), aquaporin TIP41 (TIP41-Like) and cyclophilin, in two genotypes of rice, AN Cambará and BRS Querência, with different levels of soil moisture (20%, 10% and recovery) in the vegetative (V5) and reproductive stages (period preceding flowering). Currently, there are different softwares that perform stability analyses and define the most suitable reference genes for a particular study. In this study, we used five different methods: geNorm, BestKeeper, ΔCt method, NormFinder and RefFinder. The results indicate that UBC-E2 and UBQ5 can be used as reference genes in all samples and softwares evaluated. The genes ß-tubulin and eIF-4α, traditionally used as reference genes, along with GAPDH, presented lower stability values. The gene expression of basic leucine zipper (bZIP23 and bZIP72) was used to validate the selected reference genes, demonstrating that the use of an inappropriate reference can induce erroneous results.

Identification of a novel microRNA, miR-4449, as a potential blood based marker in multiple myeloma.

BACKGROUND: miRNAs act in diverse biological processes including development, cell growth, apoptosis, and hematopoiesis, suggesting their role in cancer. METHODS: We examined the miRNAs perturbed in CD138+ primary multiple myeloma (MM) cells, using microarray analysis and real-time quantitative PCR (RT-qPCR). Serum miR-4449 expression levels were detected from 71 primary MM patients and 46 healthy controls by RT-qPCR. RESULTS: Our analysis revealed up-regulation of 54 and down-regulation of 28 miRNAs in MM subjects compared to healthy controls. miR-4449 has not been reported in MM. It was found that the relative expression of bone marrow miR-4449 in MM patients (2.14±1.42) was higher than that in healthy controls (0.815±0.165) (U=8, p=0.0093). The relative expression of serum miR-4449 in MM patients (2.11±2.10) was significantly higher than that in healthy controls (0.357±0.235) (U=374, p<0.0001) and was significantly correlated with ß2M, λ light and κ light chain concentration (r=0.480, p=0.0003; r=0.560, p<0.0001; r=0.560, p<0.0001), but not correlated with the lactate dehydrogenase (LDH) concentration (r=0.247, p=0.0611). The area under the curve (AUC) of the receiver-operating characteristics (ROC) curve of serum miR-4449 was 0.885 (95% CI, 0.826-0.945), which is higher than for other markers. Combining miR-4449, λ light chain, and ß2M together, the sensitivity was highest compared with λ light chain or ß2M alone, or combined. CONCLUSIONS: The expression levels of serum miR-4449 in MM patients were significantly higher than in healthy controls, suggesting that it may prove to be useful in the auxiliary diagnosis of MM.

Evaluation of urine for Leishmania infantum DNA detection by real-time quantitative PCR.

The availability of some sorts of biological samples which require noninvasive collection methods has led to an even greater interest in applying molecular biology on visceral leishmaniasis (VL) diagnosis, since these samples increase the safety and comfort of both patients and health professionals. In this context, this work aimed to evaluate the suitability of the urine as a specimen for Leishmania infantum kinetoplast DNA detection by real-time quantitative PCR (qPCR). Subsequent to the reproducibility analysis, the detection limit of the qPCR assay was set at 5fg (~0.025 parasites) per µL of urine. From the comparative analysis performed with a set of diagnostic criteria (serological and molecular reference tests), concordance value of 96.08% was obtained (VL-suspected and HIV/AIDS patients, n=51) (P>0.05). Kappa coefficient (95% CI) indicated a good agreement between the test and the set of diagnostic criteria (k=0.778±0.151). The detection of Leishmania DNA in urine by qPCR was possible in untreated individuals, and in those with or without suggestive renal impairment. Fast depletion of the parasite's DNA in urine after treatment (from one dose of meglumine antimoniate) was suggested by negative qPCR results, thus indicating it as a potential alternative specimen to follow up the efficacy of therapeutic approaches. Even when evaluated in a clinically heterogeneous set of patients, the urine showed good prospect as sample for VL diagnosis by qPCR, also indicating a good negative predictive value for untreated suspected patients.

Isolation and expression of two polyketide synthase genes from Trichoderma harzianum 88 during mycoparasitism

Abstract Metabolites of mycoparasitic fungal species such as Trichoderma harzianum 88 have important biological roles. In this study, two new ketoacyl synthase (KS) fragments were isolated from cultured Trichoderma harzianum 88 mycelia using degenerate primers and analysed using a phylogenetic tree. The gene fragments were determined to be present as single copies in Trichoderma harzianum 88 through southern blot analysis using digoxigenin-labelled KS gene fragments as probes. The complete sequence analysis in formation of pksT-1 (5669 bp) and pksT-2 (7901 bp) suggests that pksT-1 exhibited features of a non-reducing type I fungal PKS, whereas pksT-2 exhibited features of a highly reducing type I fungal PKS. Reverse transcription polymerase chain reaction indicated that the isolated genes are differentially regulated in Trichoderma harzianum 88 during challenge with three fungal plant pathogens, which suggests that they participate in the response of Trichoderma harzianum 88 to fungal plant pathogens. Furthermore, disruption of the pksT-2 encoding ketosynthase–acyltransferase domains through Agrobacterium -mediated gene transformation indicated that pksT-2 is a key factor for conidial pigmentation in Trichoderma harzianum 88.

Isolation and expression of two polyketide synthase genes from Trichoderma harzianum 88 during mycoparasitism

Metabolites of mycoparasitic fungal species such as Trichoderma harzianum 88 have important biological roles. In this study, two new ketoacyl synthase (KS) fragments were isolated from cultured Trichoderma harzianum 88 mycelia using degenerate primers and analysed using a phylogenetic tree. The gene fragments were determined to be present as single copies in Trichoderma harzianum 88 through southern blot analysis using digoxigenin-labelled KS gene fragments as probes. The complete sequence analysis in formation of pksT-1 (5669 bp) and pksT-2 (7901 bp) suggests that pksT-1 exhibited features of a non-reducing type I fungal PKS, whereas pksT-2 exhibited features of a highly reducing type I fungal PKS. Reverse transcription polymerase chain reaction indicated that the isolated genes are differentially regulated in Trichoderma harzianum 88 during challenge with three fungal plant pathogens, which suggests that they participate in the response of Trichoderma harzianum 88 to fungal plant pathogens. Furthermore, disruption of the pksT-2 encoding ketosynthaseacyltransferase domains through Agrobacterium -mediated gene transformation indicated that pksT-2 is a key factor for conidial pigmentation in Trichoderma harzianum 88.(AU)

Isolation and expression of two polyketide synthase genes from Trichoderma harzianum 88 during mycoparasitism.

Metabolites of mycoparasitic fungal species such as Trichoderma harzianum 88 have important biological roles. In this study, two new ketoacyl synthase (KS) fragments were isolated from cultured Trichoderma harzianum 88 mycelia using degenerate primers and analysed using a phylogenetic tree. The gene fragments were determined to be present as single copies in Trichoderma harzianum 88 through southern blot analysis using digoxigenin-labelled KS gene fragments as probes. The complete sequence analysis in formation of pksT-1 (5669bp) and pksT-2 (7901bp) suggests that pksT-1 exhibited features of a non-reducing type I fungal PKS, whereas pksT-2 exhibited features of a highly reducing type I fungal PKS. Reverse transcription polymerase chain reaction indicated that the isolated genes are differentially regulated in Trichoderma harzianum 88 during challenge with three fungal plant pathogens, which suggests that they participate in the response of Trichoderma harzianum 88 to fungal plant pathogens. Furthermore, disruption of the pksT-2 encoding ketosynthase-acyltransferase domains through Agrobacterium-mediated gene transformation indicated that pksT-2 is a key factor for conidial pigmentation in Trichoderma harzianum 88.

Decreased expression of MicroRNA-200 family in human breast cancer is associated with lymph node metastasis.

OBJECTIVES: MicroRNA-200 family (miR-200f) has been consistently reported to be deregulated and modulate the metastatic process in multiple cancers. In the present study, we detected the expression of miR-200f in breast cancer (BC) tissue and explored its relationships with clinicopathological characteristics, especially with lymph node metastasis. METHODS: Expression levels of miR-200a, miR-200b, miR-200c, miR-141, and miR-429 in 99 pairs of BC tissues and adjacent normal tissues were measured by real-time quantitative PCR. The correlation between miR-200f level and multiple clinicopathological factors was then examined by Mann-Whitney test, ANOVA, and operating characteristic (ROC) analysis. RESULTS: All members of the miR-200f were down-regulated in BC tissue compared with that in normal adjacent tissue; miR-200a, miR-200b, and miR-200c were highly decreased (p < 0.05), while the differences of miR-141 and miR-429 between patients and the control group were not statistically significant. Furthermore, all five members were found to be distinctly decreased with the incidence of lymph node metastasis (p < 0.05); When the patients were divided into three groups according to the number of lymph node metastasis (0; 1-3; ≥4), a gradual decrease of miR-200f expression was observed with the increasing number of lymph node metastasis; ROC revealed that miR-200b can differentiate patients with lymph node metastasis from those without lymph node metastasis. CONCLUSION: These observations imply that the down-regulation of miR-200f in human BC is associated with an invasive phenotype, and miR-200b may be useful to estimate the likelihood of the presence of pathologically positive lymph nodes.

Gene Expression Profile in Peripheral Blood Cells of Friedreich Ataxia Patients.

Friedreich ataxia (FRDA) is the most common autosomal recessive ataxia characterized by a combination of neurological involvement, cardiomyopathy, and skeletal and glucose metabolism disturbances. FRDA is caused by mutations in FXN gene that results in reduction of mRNA and protein levels of frataxin. Previous microarray and real-time quantitative PCR (qPCR) studies showed that the downregulation of FXN is associated with a complex gene expression profile. However, these studies showed a wide variability in the subset of genes with altered expression among tissues and models. Genes differentially expressed in peripheral blood cells (PBC) could potentially help in the understanding of FRDA pathophysiology and also function as reliable disease biomarkers obtained from an easily accessible tissue, which could have implications in clinical practice. This study aimed to validate by qPCR the expression of 26 genes, revealed as differentially expressed by other studies, using peripheral blood cells (PBC) of 11 FRDA patients compared to 11 healthy controls. We found a robust downregulation of FXN, but no statistically significant differences were found between FRDA and controls for the remaining genes. Except for FXN, our study did not find a differential gene expression profile in PBC of FRDA patients and a reliable gene expression profile biomarker in a clinical relevant and noninvasive tissue remains unclear.

Characterization and expression analysis of WOX2 homeodomain transcription factor in Aegilops tauschii.

The WUSCHEL (WUS)-related homeobox (WOX) gene family coordinates transcription during the early phases of embryogenesis. In this study, a putative WOX2 homolog was isolated and characterized from Aegilops tauschii, the donor of D genome of Triticum aestivum. The sequence consisted of 2045 bp, and contained an open reading frame (ORF), encoded 322 amino acids. The predicted protein sequence contained a highly conserved homeodomain and the WUS-box domain, which is present in some members of the WOX protein family. The full-length ORF was subcloned into prokaryotic expression vector pET-30a, and an approximately 34-kDa protein was expressed in Escherichia coli BL21 (DE3) cells with IPTG induction. The molecular mass of the expressed protein was identical to that predicted by the cDNA sequence. Phylogenetic analysis suggested that Ae. tauschii WOX2 is closely related to the rice and maize orthologs. Quantitative PCR analysis showed that WOX2 from Ae. tauschii was primarily expressed in the seeds; transcription increased during seed development and declined after the embryos matured, suggesting that WOX2 is associated with embryo development in Ae. tauschii.

Development and validation of an efficient real-time quantitative reverse transcription polymerase chain reaction assay of Chinese goose CD4 and CD8

Background: CD4+ T cells, which are often referred as T-helper cells, play a central role through secreting various cytokinesto enhance immune defense to pathogen. CD8+ T cells, which are called cytotoxic T lymphocytes (CTLs), provide potentdefenses against virus infection and intracellular pathogens by killing the targets cells directly. In our previous researches,the conventional and semi-quantitative PCR were used to detect the goose CD4 and CD8α. However, the semi-quantitativeRT-PCR only detect the relative amount of gene transcription. Quantitative PCR assay was more sensitive than conventionalPCR assay, and quantitative PCR assay has a lower limit of sensitivity.Materials, Methods & Results: Contrast to conventional assays, the detection of amplicons by quantitative RT-PCR couldbe visualized as the amplifi cation progressed. This effect has provided a great deal of insight into the kinetics of the reaction and it is the foundation of kinetic of real-time qPCR. The analysis of gene transcription by qPCR has proven to bean attractive method due to its potential for increasing laboratory throughput, simultaneous processing of several samplesas well as more reliable instrumentation. With those in mind, the real-time quantitative reverse transcription PCR (qRTPCR) methods for the detection of goose CD4 and CD8α transcripts were reported here for the fi rst time. With this assay,it is possible to carry out a rapid quantitative analysis of goose CD4 and CD8α transcripts over a wide linear range, withan unknown template.CD8 is expressed on the membrane of T cells either as an αα-homodimer or αβ-heterodimer. Sinceboth forms of CD8 have α chain, the transcription levels of CD8 can be monitored by detecting CD8α mRNA expression.Assays were based on the DNA sequence of goose CD4 [GenBank: JX902315], CD8α [GenBank: KC476104], and β-actin[GenBank: M26111]. qPCR was carried out in quadruplicates in a total volume of 20 µL containing...

Development and validation of an efficient real-time quantitative reverse transcription polymerase chain reaction assay of Chinese goose CD4 and CD8

Background: CD4+ T cells, which are often referred as T-helper cells, play a central role through secreting various cytokinesto enhance immune defense to pathogen. CD8+ T cells, which are called cytotoxic T lymphocytes (CTLs), provide potentdefenses against virus infection and intracellular pathogens by killing the targets cells directly. In our previous researches,the conventional and semi-quantitative PCR were used to detect the goose CD4 and CD8α. However, the semi-quantitativeRT-PCR only detect the relative amount of gene transcription. Quantitative PCR assay was more sensitive than conventionalPCR assay, and quantitative PCR assay has a lower limit of sensitivity.Materials, Methods & Results: Contrast to conventional assays, the detection of amplicons by quantitative RT-PCR couldbe visualized as the amplifi cation progressed. This effect has provided a great deal of insight into the kinetics of the reaction and it is the foundation of kinetic of real-time qPCR. The analysis of gene transcription by qPCR has proven to bean attractive method due to its potential for increasing laboratory throughput, simultaneous processing of several samplesas well as more reliable instrumentation. With those in mind, the real-time quantitative reverse transcription PCR (qRTPCR) methods for the detection of goose CD4 and CD8α transcripts were reported here for the fi rst time. With this assay,it is possible to carry out a rapid quantitative analysis of goose CD4 and CD8α transcripts over a wide linear range, withan unknown template.CD8 is expressed on the membrane of T cells either as an αα-homodimer or αβ-heterodimer. Sinceboth forms of CD8 have α chain, the transcription levels of CD8 can be monitored by detecting CD8α mRNA expression.Assays were based on the DNA sequence of goose CD4 [GenBank: JX902315], CD8α [GenBank: KC476104], and β-actin[GenBank: M26111]. qPCR was carried out in quadruplicates in a total volume of 20 µL containing...(AU)

Molecular cloning and functional analysis of the fatty acid-binding protein (Sp-FABP) gene in the mud crab (Scylla paramamosain).

Intracellular fatty acid-binding proteins (FABPs) are multifunctional cytosolic lipid-binding proteins found in vertebrates and invertebrates. In this work, we used RACE to obtain a full-length cDNA of Sp-FABP from the mud crab Scylla paramamosain. The open reading frame of the full length cDNA (886 bp) encoded a 136 amino acid polypeptide that showed high homology with related genes from other species. Real-time quantitative PCR identified variable levels of Sp-FABP transcripts in epidermis, eyestalk, gill, heart, hemocytes, hepatopancreas, muscle, ovary, stomach and thoracic ganglia. In ovaries, Sp-FABP expression increased gradually from stage I to stage IV of development and decreased in stage V. Sp-FABP transcripts in the hepatopancreas and hemocytes were up-regulated after a bacterial challenge with Vibrio alginnolyficus. These results suggest that Sp-FABP may be involved in the growth, reproduction and immunity of the mud crab.

Identification of chilling-responsive transcripts in peanut (Arachis hypogaea L. )

To isolate differentially expressed peanut genes responsive to chilling, a suppression subtractive hybridization (SSH) cDNA library was constructed for a chilling tolerant peanut cultivar A4 with mRNAs extracted from the seeds imbibed at 2ºC and 15ºC, respectively, for 24 hrs. A total of 466 cDNA clones were sequenced, from which 193 unique transcripts (73 contigs and 120 singlets) were assembled. Of these unique transcripts, 132 (68.4 percent) were significantly similar to the sequences in GenBank non-redundant (nr) protein database, which belonged to diverse functional categories including metabolism, signal transduction, stress response, cell defense and transcriptional regulation. The remaining 61 (31.6 percent) showed no similarity to either hypothetical or known proteins. Six differentially expressed transcripts were further confirmed with real-time quantitative PCR (RT-qPCR).