Validation of reference genes for RT-qPCR normalization in common bean during biotic and abiotic stresses

Selection of reference genes is an essential consideration to increase the precision and quality of relative expression analysis by the quantitative RT-PCR method. The stability of eight expressed sequence tags was evaluated to define potential reference genes to study the differential expression of common bean target genes under biotic (incompatible interaction between common bean and fungus Colletotrichum lindemuthianum) and abiotic (drought; salinity; cold temperature) stresses. The efficiency of amplification curves and quantification cycle (C (q)) were determined using LinRegPCR software. The stability of the candidate reference genes was obtained using geNorm and NormFinder software, whereas the normalization of differential expression of target genes [beta-1,3-glucanase 1 (BG1) gene for biotic stress and dehydration responsive element binding (DREB) gene for abiotic stress] was defined by REST software. High stability was obtained for insulin degrading enzyme (IDE), actin-11 (Act11), unknown 1 (Ukn1) and unknown 2 (Ukn2) genes during biotic stress, and for SKP1/ASK-interacting protein 16 (Skip16), Act11, Tubulin beta-8 (beta-Tub8) and Unk1 genes under abiotic stresses. However, IDE and Act11 were indicated as the best combination of reference genes for biotic stress analysis, whereas the Skip16 and Act11 genes were the best combination to study abiotic stress. These genes should be useful in the normalization of gene expression by RT-PCR analysis in common bean, the most important edible legume.

Trichoderma harzianum expressed sequence tags for identification of genes with putative roles in mycoparasitism against Fusarium solani

The plant pathogen Fusarium solani causes a disease root rot of common bean (Phaseolus vulgaris) resulting in great losses of yield in irrigated areas of the Southeast and Midwest regions of Brazil. Species of the genus Trichoderma have been used in the biological control of this pathogen as an alternative to chemical control. To gain new insights into the biocontrol mechanism used by Trichoderma harzianum against the phytopathogenic fungus, Fusarium solani, we performed a transcriptome analysis using expressed sequence tags (ESTs) and quantitative real-time PCR (RT-qPCR) approaches. A cDNA library from T. harzianum mycelium (isolate ALL42) grown on cell walls of F. solani (CWFS) was constructed and analyzed. A total of 2927 high quality sequences were selected from 3845 and 37.7% were identified as unique genes. The Gene Ontology analysis revealed that the majority of the annotated genes are involved in metabolic processes (80.9%), followed by cellular process (73.7%). We tested twenty genes that encode proteins with potential role in biological control. RT-qPCR analysis showed that none of these genes were expressed when T. harzianum was challenged with itself. These genes showed different patterns of expression during in vitro interaction between T. harzianum and F. solani. (C) 2012 Elsevier Inc. All rights reserved.

Molecular Surveillance of the Newcastle Disease Virus in Domestic and Wild Birds on the North Eastern Coast and Amazon Biome of Brazil

Brazil is one of the world's largest countries with a rich diversity of wildlife, including resident and migratory wild birds, which may be natural reservoirs of the Newcastle disease virus (NDV). Because Brazil is a major global exporter of chicken meat, the emergence of such a disease may have a huge negative impact not only on the economy due to trade restrictions and embargoes, but also on the quality of life of the population. Samples were collected from 1,022 asymptomatic domestic and wild birds from the Brazilian coast and the Amazon region using tracheal/cloacal swabs and tested by RT-qPCR. The results showed 7 (0.7%) birds were positive for NDV. The positive samples were then isolated in embryonated chicken eggs and their matrix protein genes were partially sequenced, revealing a low-pathogenicity NDV. This study confirms the maintenance of the velogenic-NDV free status of Brazil.

ORAOV1 is amplified in oral squamous cell carcinoma

BACKGROUND: Oral cancer overexpressed 1 (ORAOV1) was found as a candidate oncogene in the 11q13 chromosomal region, based on its amplification and overexpression in oral cancer cell lines. Because gene amplification often leads to increased levels of gene expression, we aimed to verify the relationship between ORAOV1 gene status and mRNA expression primarily in oral squamous cell carcinoma (OSCC) by quantitative assay, correlating with clinical and pathological characteristics in patients. METHODS: Levels of ORAOV1 amplification and expression were evaluated by qPCR and RT-qPCR in OSCC cell lines and in tumor and non-tumoral surgical margins from 33 patients with OSCC. All subjects were smokers and habitual alcohol drinkers, mostly men above 40 years of age and with a single primary tumor. RESULTS: ORAOV1 exhibited increased gene expression levels as well as higher copy number in three OSCC cell lines with 11q13 amplified chromosomal region when compared with the OSCC cell line without the amplification (one-way ANOVA, P < 0.05). Weak correlation between ORAOV1 mRNA levels and DNA copy number was seen in tumor samples (Spearman, P = 0.07). Although ORAOV1 was amplified in tumor (Wilcoxon, P < 0.01), high levels of transcripts in margin did not reveal differences in comparison with tumor (Wilcoxon, P = 0.85). Aggressiveness and survival rate did not demonstrate statistical difference for both events in OSCC. CONCLUSION: The overexpression of ORAOV1 in non-tumoral margin samples can occur in the absence of amplification. The weak correlation between ORAOV1 amplification and expression in OSSC suggests that ORAOV1 expression can be regulated by mechanisms other than gene amplification. J Oral Pathol Med (2012) 41: 5460

Changes in spatial and temporal gene expression during incompatible interaction between common bean and anthracnose pathogen

Common bean, one of the most important legumes for human consumption, may have drastic reduction in yield due to anthracnose, a disease caused by the fungus Colletotrichum lindemuthianum. Rapid induction of the plant defense mechanisms is essential to establish an incompatible interaction with this pathogenic fungus. In this study, we evaluated spatial (leaves, epicotyls and hypocotyls) and temporal (24, 48, 72 and 96 hours after inoculation [HAI]) relative expression (RE) of 12 defense-related transcripts selected from previously developed ESTs libraries, during incompatible interaction between the resistant common bean genotype SEL 1308 and the avirulent anthracnose pathogen race 73, using real time quantitative RT-PCR (RT-qPCR) analysis. All selected transcripts, including the ones coding for pathogenesis-related (PR) proteins (PR1a, PR1b, PR2, and PR16a and PR16b) were differentially regulated upon pathogen inoculation. The expression levels of these transcripts were dependent on the tissue and time post inoculation. This study contributes to a better understanding of the kinetics of induced defenses against a fungal pathogen of common bean and may be used as a base line to study defenses against a broad range of pathogens including bacteria as well as non-host resistance. (C) 2012 Elsevier GmbH. All rights reserved.

Molecular measurement of BCR-ABL transcript variations in chronic myeloid leukemia patients in cytogenetic remission

Abstract Background The monitoring of BCR-ABL transcript levels by real-time quantitative polymerase chain reaction (RT-qPCR) has become important to assess minimal residual disease (MRD) and standard of care in the treatment of chronic myeloid leukemia (CML). In this study, we performed a prospective, sequential analysis using RT-qPCR monitoring of BCR-ABL gene rearrangements in blood samples from 91 CML patients in chronic phase (CP) who achieved complete cytogenetic remission (CCyR) and major molecular remission (MMR) throughout imatinib treatment. Methods The absolute level of BCR-ABL transcript from peripheral blood was serially measured every 4 to 12 weeks by RT-qPCR. Only level variations > 0.5%, according to the international scale, was considered positive. Sequential cytogenetic analysis was also performed in bone marrow samples from all patients using standard protocols. Results Based on sequential analysis of BCR-ABL transcripts, the 91 patients were divided into three categories: (A) 57 (62.6%) had no variation on sequential analysis; (B) 30 (32.9%) had a single positive variation result obtained in a single sample; and (C) 4 (4.39%) had variations of BCR-ABL transcripts in at least two consecutive samples. Of the 34 patients who had elevated levels of transcripts (group B and C), 19 (55.8%) had a < 1% of BCR-ABL/BCR ratio, 13 (38.2%) patients had a 1% to 10% increase and 2 patients had a >10% increase of RT-qPCR. The last two patients had lost a CCyR, and none of them showed mutations in the ABL gene. Transient cytogenetic alterations in Ph-negative cells were observed in five (5.5%) patients, and none of whom lost CCyR. Conclusions Despite an increase levels of BCR-ABL/BCR ratio variations by RT-qPCR, the majority of CML patients with MMR remained in CCyR. Thus, such single variations should neither be considered predictive of subsequent failure and nor an indication for altering imatinib dose or switching to second generation therapy. Changing of imatinib on the basis of BCR-ABL/BCR% sustained increase and mutational studies is a prudent approach for preserving other therapeutic options in imatinib-resistant patients.

Identification of differentially expressed genes from Trichoderma harzianum during growth on cell wall of Fusarium solani as a tool for biotechnological application

Background: The species of T. harzianum are well known for their biocontrol activity against many plant pathogens. However, there is a lack of studies concerning its use as a biological control agent against F. solani, a pathogen involved in several crop diseases. In this study, we have used subtractive library hybridization (SSH) and quantitative real-time PCR (RT-qPCR) techniques in order to explore changes in T. harzianum genes expression during growth on cell wall of F. solani (FSCW) or glucose. RT-qPCR was also used to examine the regulation of 18 genes, potentially involved in biocontrol, during confrontation between T. harzianum and F. solani. Results: Data obtained from two subtractive libraries were compared after annotation using the Blast2GO suite. A total of 417 and 78 readable EST sequence were annotated in the FSCW and glucose libraries, respectively. Functional annotation of these genes identified diverse biological processes and molecular functions required during T. harzianum growth on FSCW or glucose. We identified various genes of biotechnological value encoding to proteins which function such as transporters, hydrolytic activity, adherence, appressorium development and pathogenesis. Fifteen genes were up-regulated and sixteen were down-regulated at least at one-time point during growth of T. harzianum in FSCW. During the confrontation assay most of the genes were up-regulated, mainly after contact, when the interaction has been established. Conclusions: This study demonstrates that T. harzianum expressed different genes when grown on FSCW compared to glucose. It provides insights into the mechanisms of gene expression involved in mycoparasitism of T. harzianum against F. solani. The identification and evaluation of these genes may contribute to the development of an efficient biological control agent.

Transcriptional effects of 1,25 dihydroxyvitamin D3 physiological and supra-physiological concentrations in breast cancer organotypic culture

Background Vitamin D transcriptional effects were linked to tumor growth control, however, the hormone targets were determined in cell cultures exposed to supra physiological concentrations of 1,25(OH)2D3 (50-100nM). Our aim was to evaluate the transcriptional effects of 1,25(OH)2D3 in a more physiological model of breast cancer, consisting of fresh tumor slices exposed to 1,25(OH)2D3 at concentrations that can be attained in vivo. Methods Tumor samples from post-menopausal breast cancer patients were sliced and cultured for 24 hours with or without 1,25(OH)2D3 0.5nM or 100nM. Gene expression was analyzed by microarray (SAM paired analysis, FDR≤0.1) or RT-qPCR (p≤0.05, Friedman/Wilcoxon test). Expression of candidate genes was then evaluated in mammary epithelial/breast cancer lineages and cancer associated fibroblasts (CAFs), exposed or not to 1,25(OH)2D3 0.5nM, using RT-qPCR, western blot or immunocytochemistry. Results 1,25(OH)2D3 0.5nM or 100nM effects were evaluated in five tumor samples by microarray and seven and 136 genes, respectively, were up-regulated. There was an enrichment of genes containing transcription factor binding sites for the vitamin D receptor (VDR) in samples exposed to 1,25(OH)2D3 near physiological concentration. Genes up-modulated by both 1,25(OH)2D3 concentrations were CYP24A1, DPP4, CA2, EFTUD1, TKTL1, KCNK3. Expression of candidate genes was subsequently evaluated in another 16 samples by RT-qPCR and up-regulation of CYP24A1, DPP4 and CA2 by 1,25(OH)2D3 was confirmed. To evaluate whether the transcripitonal targets of 1,25(OH)2D3 0.5nM were restricted to the epithelial or stromal compartments, gene expression was examined in HB4A, C5.4, SKBR3, MDA-MB231, MCF-7 lineages and CAFs, using RT-qPCR. In epithelial cells, there was a clear induction of CYP24A1, CA2, CD14 and IL1RL1. In fibroblasts, in addition to CYP24A1 induction, there was a trend towards up-regulation of CA2, IL1RL1, and DPP4. A higher protein expression of CD14 in epithelial cells and CA2 and DPP4 in CAFs exposed to 1,25(OH)2D3 0.5nM was detected. Conclusions In breast cancer specimens a short period of 1,25(OH)2D3 exposure at near physiological concentration modestly activates the hormone transcriptional pathway. Induction of CYP24A1, CA2, DPP4, IL1RL1 expression appears to reflect 1,25(OH)2D3 effects in epithelial as well as stromal cells, however, induction of CD14 expression is likely restricted to the epithelial compartment.

Unveiling novel genes upregulated by both rhBMP2 and rhBMP7 during early osteoblastic transdifferentiation of C2C12 cells

Abstract Findings We set out to analyse the gene expression profile of pre-osteoblastic C2C12 cells during osteodifferentiation induced by both rhBMP2 and rhBMP7 using DNA microarrays. Induced and repressed genes were intercepted, resulting in 1,318 induced genes and 704 repressed genes by both rhBMP2 and rhBMP7. We selected and validated, by RT-qPCR, 24 genes which were upregulated by rhBMP2 and rhBMP7; of these, 13 are related to transcription (Runx2, Dlx1, Dlx2, Dlx5, Id1, Id2, Id3, Fkhr1, Osx, Hoxc8, Glis1, Glis3 and Cfdp1), four are associated with cell signalling pathways (Lrp6, Dvl1, Ecsit and PKCδ) and seven are associated with the extracellular matrix (Ltbp2, Grn, Postn, Plod1, BMP1, Htra1 and IGFBP-rP10). The novel identified genes include: Hoxc8, Glis1, Glis3, Ecsit, PKCδ, LrP6, Dvl1, Grn, BMP1, Ltbp2, Plod1, Htra1 and IGFBP-rP10. Background BMPs (bone morphogenetic proteins) are members of the TGFβ (transforming growth factor-β) super-family of proteins, which regulate growth and differentiation of different cell types in various tissues, and play a critical role in the differentiation of mesenchymal cells into osteoblasts. In particular, rhBMP2 and rhBMP7 promote osteoinduction in vitro and in vivo, and both proteins are therapeutically applied in orthopaedics and dentistry. Conclusion Using DNA microarrays and RT-qPCR, we identified both previously known and novel genes which are upregulated by rhBMP2 and rhBMP7 during the onset of osteoblastic transdifferentiation of pre-myoblastic C2C12 cells. Subsequent studies of these genes in C2C12 and mesenchymal or pre-osteoblastic cells should reveal more details about their role during this type of cellular differentiation induced by BMP2 or BMP7. These studies are relevant to better understanding the molecular mechanisms underlying osteoblastic differentiation and bone repair.

Molecular surveillance of the Newcastle disease virus in domestic and wild birds on the North Eastern Coast and Amazon biome of Brazil

Brazil is one of the world's largest countries with a rich diversity of wildlife, including resident and migratory wild birds, which may be natural reservoirs of the Newcastle disease virus (NDV). Because Brazil is a major global exporter of chicken meat, the emergence of such a disease may have a huge negative impact not only on the economy due to trade restrictions and embargoes, but also on the quality of life of the population. Samples were collected from 1,022 asymptomatic domestic and wild birds from the Brazilian coast and the Amazon region using tracheal/cloacal swabs and tested by RT-qPCR. The results showed 7 (0.7%) birds were positive for NDV. The positive samples were then isolated in embryonated chicken eggs and their matrix protein genes were partially sequenced, revealing a low-pathogenicity NDV. This study confirms the maintenance of the velogenic-NDV free status of Brazil.

CLONING AND EXPRESSION ANALYSIS OF ALLOGRAFT INFLAMMATORY FACTOR TYPE 1 IN COELOMOCYTES OF ANTARCTIC SEA URCHIN (STERECHINUS NEUMAYERI)

We have cloned and characterized for the first time an allograft inflammatory factor 1 (Sn-AIF-1) from the Antarctic sea urchin. We report the cloning of Sn-AIF-1 cDNA and the characterization of its expression in coelomocytes after a bacterial challenge. The cDNA Sn-AIF-1 has a size of 608 bp and encodes a polypeptide of 151 aa. The deduced amino acid sequence has a putative size of 17.430 Da, an isoelectric point of 4.92, and shows 2 elongation factor handlike motifs that normally bind calcium ions. BLAST analysis revealed close matches with other known AIF-1. The deduced amino acid sequence of Sn-AIF-1 showed high homology with AIF-1 in vertebrates such as fish, mice, and humans; and in the case of invertebrates, the major degree of identity (55%) was with a predicted sequence of the purple sea urchin AIF-1, and 52% corresponded to a sponge. Expression of Sn-AIF-1 mRNA was analyzed by qPCR. Sn-AIF-1 mRNA expression was measured from coelomocytes after a bacterial challenge using RT-PCR and revealed that the gene was upregulated after 24 h. Sn-AIF-1 could participate in the inflammatory response, particularly in the activation of coelomocytes and their survival.

Comparison of fast-track diagnostics respiratory pathogens multiplex real-time RT-PCR assay with in-house singleplex assays for comprehensive detection of human respiratory viruses

Fast-track Diagnostics respiratory pathogens (FTDRP) multiplex real-time RT-PCR assay was compared with in-house singleplex real-time RT-PCR assays for detection of 16 common respiratory viruses. The FTDRP assay correctly identified 26 diverse respiratory virus strains, 35 of 41 (85%) external quality assessment samples spiked with cultured virus and 232 of 263 (88%) archived respiratory specimens that tested positive for respiratory viruses by in-house assays. Of 308 prospectively tested respiratory specimens selected from children hospitalized with acute respiratory illness, 270 (87.7%) and 265 (86%) were positive by FTDRP and in-house assays for one or more viruses, respectively, with combined test results showing good concordance (K=0.812, 95% CI = 0.786-0.838). Individual FTDRP assays for adenovirus, respiratory syncytial virus and rhinovirus showed the lowest comparative sensitivities with in-house assays, with most discrepancies occurring with specimens containing low virus loads and failed to detect some rhinovirus strains, even when abundant. The FTDRP enterovirus and human bocavirus assays appeared to be more sensitive than the in-house assays with some specimens. With the exceptions noted above, most FTDRP assays performed comparably with in-house assays for most viruses while offering enhanced throughput and easy integration by laboratories using conventional real-time PCR instrumentation. Published by Elsevier B.V.

Diagnosis of rabies and eastern and western equine viral encephalitides in equids by multiplex Hemi-Nested RT-PCR technique

Financial support: CNPq and Pasteur Institute of São Paulo