RT-PCR Detection (rt-pcr + detection)

Distribution by Scientific Domains


Selected Abstracts


RT-PCR Detection of Odontoglossum ringspot virus, Cymbidium mosaic virus and Tospoviruses and Association of Infections with Leaf-Yellowing Symptoms in Phalaenopsis

JOURNAL OF PHYTOPATHOLOGY, Issue 5 2008
K. Yamane
Abstract Leaf-yellowing symptoms in Phalaenopsis are most important effects in its commercial production in Japan and their cause has not yet been clarified. In the present study, Odontoglossum ringspot virus (ORSV), Cymbidium mosaic virus (CymMV) and tospoviruses were evaluated by reverse transcription-polymerase chain reaction (RT-PCR) in Phalaenopsis showing leaf-yellowing symptoms. Ring, spot, mosaic, necrosis and other symptom types were observed in 42%, 24%, 25%, 16% and 44% of the tested plants respectively. ORSV was detected in 55% of the plants, particularly in 35 of 42 plants with ring symptoms. CymMV was detected in 34% of the plants, particularly in 18 of 25 plants with mosaic symptoms. Plants co-infected with both viruses tended to show severe symptoms. In spite of systemic infection by inoculation of ORSV, no clear ring symptoms were observed in any plant for 6 months. Symptoms of leaf-yellowing were significantly reduced after 3 months. These results suggest that the symptoms can be associated with the viruses but occurred and alleviated over time and by changes in the environmental conditions. No tospoviruses were detected in 70 tested plants. ORSV and CymMV were simultaneously detected by RT-PCR using reported primers for ORSV and newly designed primers for CymMV using an efficient direct tube RNA extraction technique providing more cost-effective RT-PCR screening. No viruses were detected by RT-PCR in several plants showing spot or ring symptoms, suggesting the presence of other causal agents related to these symptoms. [source]


MBP-1 is efficiently encoded by an alternative transcript of the ENO1 gene but post-translationally regulated by proteasome-dependent protein turnover

FEBS JOURNAL, Issue 20 2010
Jrhau Lung
The c-myc promoter-binding protein-1 (MBP-1) is a transcriptional suppressor of tumorigenesis and thought to be the product of alternative translation initiation of the ,-enolase (ENO1) transcript. In the present study, we cloned a 2552-bp novel cDNA with a putative coding sequence of MBP-1 and functionally examined its ability to encode the MBP-1 protein. Similarly to ENO1, the obtained MBP-1 was widely and differentially expressed in a variety of normal tissues and cancer cells. Experiments using MBP-1 promoter-driven luciferase reporter assays, biochemical cell fractionation followed by RT-PCR detection of the cytoplasmic mRNA, and transcription/translation-coupled reactions, consistently demonstrated that this novel transcript was alternatively transcribed from intron III of the ENO1 gene and was feasible for MBP-1 production. Hypoxia treatments significantly increased the transcriptional activation of the MBP-1 gene. Blocking the proteasomal degradation by MG132 stabilized the MBP-1 protein in cells. Compared with the translation efficiency for production of the MBP-1 protein, the MBP-1 transcript was 17.8 times more efficient than the ENO1 transcript. Thus, we suggest that this newly discovered transcript is a genuine template for the protein synthesis of MBP-1 in cells, and optimal expression of this gene in tumors may lead to effective clinical therapies for cancers. [source]


Real-time RT-PCR detection of CK19, CK7 and MUC1 mRNA for diagnosis of lymph node micrometastases in non small cell lung carcinoma

INTERNATIONAL JOURNAL OF CANCER, Issue 5 2005
Pierre Saintigny
Abstract Metastatic lymph nodes (LNs) are the major prognostic factor in resected non small cell lung carcinoma (NSCLC). However, almost 50% of pN0 patients relapse, suggesting metastatic cells undetected by current staging procedures. A combination of markers [cytokeratins 19 and 7 (CK19, CK7) and mucin type 1 (MUC1) mRNAs] was therefore evaluated by real-time RT-PCR in order to detect occult cancer cells. Forty-three NSCLC tumor samples, 4 micrometastatic, 6 metastatic and 84 histologically negative mediastinal LNs from 19 patients with NSCLC were evaluated as well as blood mononuclear cells from 29 healthy volunteers and 17 benign LNs. When tested on cell lines, RT-PCR was particularly efficient for evaluation of CK19, CK7 and MUC1 mRNA expression. All tumor samples were positive for at least 1 marker and 74% of samples were positive for all 3 markers. CK7 and CK19 mRNA were not detected in benign LN and blood cells from healthy donors in contrast with MUC1 mRNA. Only CK7 and CK19 mRNA were therefore used for evaluation of mediastinal LNs: the 6 histologically metastatic and the 4 micrometastatic LNs were positive for at least one marker. Among the 84 histologically negative LNs, 6 (7%) were positive for at least one marker, potentially changing the stage of 2 out of 19 patients. In conclusion, in our feasibility study, parallel molecular detection of CK19 and CK7 mRNA can be considered a specific diagnostic tool for the assessment of microscopic lymphatic spread. Its prognostic impact remains to be evaluated in a prospective study. © 2005 Wiley-Liss, Inc. [source]


Detection of Circulating Melanoma Cells by RT-PCR Amplification of Three Different Melanocyte-Specific mRNAs in a Mouse Model

PIGMENT CELL & MELANOMA RESEARCH, Issue 3 2000
KATSUHIKO TSUKAMOTO
Three different melanocyte-specific mRNAs are studied as potential markers for circulating melanoma cells in the serum of mice inoculated subcutaneously with B16F10 melanoma cells. These three mRNAs encode tyrosinase, tyrosinase related protein-2 (TRP-2) and Pmel17, proteins that are essential for the synthesis of melanin and are expressed specifically in melanocytes. We used reverse-transcription polymerase chain reaction (RT-PCR) to detect these three different melanocyte-specific mRNAs in the sera of B16F10 bearing mice. Since melanocytes would not normally be present in the blood, the detection of those transcripts should indicate the presence of circulating melanoma cells. RT-PCR detection of all three mRNAs was highly sensitive and specific. Our in vitro studies show that as few as 10 melanoma cells can be detected in 125 ,l blood and that in vivo, melanoma cells can be detected in blood samples from B16F10 melanoma bearing mice. Of these three mRNAs, Pmel17 mRNA is the most sensitive marker for detecting circulating melanoma cells compared with tyrosinase mRNA and TRP-2 mRNA. Moreover, this mouse model might be useful for basic research of malignant melanoma patients with haematogenous metastasis. [source]


Distinct Mechanism of Small-for-Size Fatty Liver Graft Injury,Wnt4 Signaling Activates Hepatic Stellate Cells

AMERICAN JOURNAL OF TRANSPLANTATION, Issue 5 2010
Q. Cheng
In this study, we aimed to investigate the significance of hepatic stellate cells (HSCs) activation in small-for-size fatty liver graft injury and to explore the underlying molecular mechanism in a rat liver transplantation model. A rat orthotopic liver transplantation model using fatty grafts (40% of fatty changes) and cirrhotic recipients was applied. Intragraft gene expression profiles, ultrastructure features and HSCs activation were compared among the rats received different types of grafts (whole vs. small-for-size, normal vs. fatty). The distinct molecular signature of small-for-size fatty graft injury was identified by cDNA microarray screening and confirmed by RT-PCR detection. In vitro functional studies were further conducted to investigate the direct effect of specific molecular signature on HSCs activation. HSCs activation was predominantly present in small-for-size fatty grafts during the first 2 weeks after transplantation, and was strongly correlated with progressive hepatic sinusoidal damage and significant upregulation of intragraft Wnt4 signaling pathway. In vitro suppression of Wnt4 expression could inhibit HSC activation directly. In conclusion, upregulation of Wnt4 signaling led to direct HSC activation and subsequently induced small-for-size fatty liver grafts injury. Discovery of this distinct mechanism may lay the foundation for prophylactic treatment for marginal graft injury in living donor liver transplantation. [source]