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Quantitative Real Time PCR (quantitative + real_time_pcr)
Selected AbstractsOriginal article: The expression of CFL1 and N-WASP in esophageal squamous cell carcinoma and its correlation with clinicopathological featuresDISEASES OF THE ESOPHAGUS, Issue 6 2010Wei-Sen Wang SUMMARY Cofilin1 (CFL1) is an actin-modulating protein, which belongs to the ADF/Cofilin family. Neural Wiskott,Aldrich syndrome protein (N-WASP) is the key regulator of the actin cytoskeleton, a member of Wiskott-Aldrich syndrome protein family. They have been suggested to be involved in cancer cell invasion and metastasis. In this study, the expression patterns of CFL1 and N-WASP in normal esophageal mucosa and esophageal squamous cell carcinoma (ESCC) and their correlation with clinical characteristics were investigated. Immunohistochemical staining showed that CFL1 was expressed in nuclear and cytoplasm of cancer cells. However, N-WASP was mainly found in the cytoplasm of the cancer cells. There were significant evidences that proved that CFL1 is correlated with clinicopathological factors in ESCC, such as infiltration depth, lymph node metastasis and pathological staging (P < 0.05). It is also proved that N-WASP is related to lymph node metastasis and pathological staging in ESCC (P < 0.05). Kaplan,Meier analysis showed that there was no correlation between CFL1 and N-WASP protein expression and survival (P > 0.05). Moreover, the mRNA expression of CFL1 and N-WASP was detected by quantitative real time PCR in 70 tissue specimens. The results showed that CFL1 mRNA level was over-expressed in ESCC tissue (P < 0.05), while N-WASP mRNA expression level was not different between cancerous tissues and adjacent normal esophageal mucosa (P > 0.05). Also, CFL1 mRNA expression was significantly associated with regional lymph node metastasis and pathological staging (P < 0.05). Kaplan,Meier analysis showed that there was no correlation between CFL1 and N-WASP mRNA expression and survival (P > 0.05). Our findings suggested that CFL1 and N-WASP may play an important role in the tumorigenesis of ESCC, and to be the candidate novel biomarkers for the diagnosis and prognosis of ESCC. These findings may have implications for targeted therapies in patients with ESCC. [source] Gene expression analysis in absence epilepsy using a monozygotic twin designEPILEPSIA, Issue 9 2008Ingo Helbig Summary Purpose: To identify genes involved in idiopathic absence epilepsies by analyzing gene expression using a monozygotic (MZ) twin design. Methods: Genome-wide gene expression in lymphoblastoid cell lines (LCLs) was determined using microarrays derived from five discordant and four concordant MZ twin pairs with idiopathic absence epilepsies and five unaffected MZ twin pairs. Gene expression was analyzed using three strategies: discordant MZ twins were compared as matched pairs, MZ twins concordant for epilepsy were compared to control MZ twins, and a singleton design of affected versus unaffected MZ twin individuals was used irrespective of twin pairing. An overlapping gene list was generated from these analyses. Dysregulation of genes recognized from the microarray experiment was validated using quantitative real time PCR (qRT-PCR) in the twin sample and in an independent sample of 18 sporadic absence cases and 24 healthy controls. Results: Sixty-five probe sets were identified from the three combined microarray analysis strategies. Sixteen genes were chosen for validation and nine of these genes confirmed by qRT-PCR in the twin sample. Differential expression for EGR1 (an immediate early gene) and RCN2 (coding for the calcium-binding protein Reticulocalbin 2) were reconfirmed by qRT-PCR in the independent sample. Discussion: Using a unique sample of discordant MZ twins, our study identified genes with altered expression, which suggests novel mechanisms in idiopathic absence epilepsy. Dysregulation of EGR1 and RCN2 is implicated in idiopathic absence epilepsy. [source] Cloning and expression of murine enzymes involved in the salvage pathway of GDP- l -fucoseFEBS JOURNAL, Issue 1 2004GDP- l -fucose pyrophosphorylase, l -fucokinase In the salvage pathway of GDP- l -fucose, free cytosolic fucose is phosphorylated by l -fucokinase to form l -fucose-1-phosphate, which is then further converted to GDP- l -fucose in the reaction catalyzed by GDP- l -fucose pyrophosphorylase. We report here the cloning and expression of murine l -fucokinase and GDP- l -fucose pyrophosphorylase. Murine l -fucokinase is expressed as two transcripts of 3057 and 3270 base pairs, encoding proteins of 1019 and 1090 amino acids with predicted molecular masses of 111 kDa and 120 kDa respectively. Only the longer splice variant of l -fucokinase was enzymatically active when expressed in COS-7 cells. Murine GDP- l -fucose pyrophosphorylase has an open reading frame of 1773 base pairs encoding a protein of 591 amino acids with a predicted molecular mass of 65.5 kDa. GDP- l -fucose, the reaction product of GDP- l -pyrophosphorylase, was identified by HPLC and MALDI-TOF MS analysis. The tissue distribution of murine l -fucokinase and GDP- l -fucose pyrophosphorylase was investigated by quantitative real time PCR, which revealed high expression of l -fucokinase and GDP- l -fucose pyrophosphorylase in various tissues. The wide expression of both enzymes can also be observed from the large amount of data collected from a number of expressed sequence tag libraries, which indicate that not only the de novo pathway alone, but also the salvage pathway, could have a significant role in the synthesis of GDP- l -fucose in the cytosol. [source] RECK expression in osteosarcoma: correlation with matrix metalloproteinases activation and tumor invasivenessJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 5 2007Hyun-Guy Kang Abstract Osteosarcoma is a malignant tumor of bone characterized by its high metastatic potential. For the development of metastasis, activation of matrix metalloproteinases (MMPs) is required. A novel MMPs inhibitor, reversion inducing cysteine rich protein with Kazal motifs (RECK), is known to down-regulate MMPs and suppress the invasive and metastatic potential in many tumor-derived cell lines and some types of tumors. The expression of RECK and its role in tumor invasiveness have never been studied in osteosarcoma. We examined RECK mRNA expression and MMPs activation in osteosarcoma using quantitative real time PCR, gelatin zymography, invasion assay, and transfection experiments. RECK was expressed but down-regulated in osteosarcoma cells. Activation of pro-MMP-2 was observed in all samples, whereas activation of MMP-2 and pro-MMP-9 was detected in only 11% and 7% of the samples, respectively. MMP-9 was not activated in any of the samples. The level of RECK expression was inversely correlated with pro-MMP-2 activation, and overexpression of RECK by transfection resulted in decreased pro-MMP-2 activation and reduced tumor invasiveness. These findings suggest that RECK plays an important role in the invasiveness of osteosarcoma. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 25:696,702, 2007 [source] Identification and quantification of differentially expressed transcripts in in vitro-produced bovine preimplantation stage embryosMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 2 2003Dawit Tesfaye Abstract In this study, we used mRNA differential display reverse transcription polymerase chain reaction (DDRT-PCR) to analyze the mRNA expression patterns in in vitro-produced bovine 8-cell, 16-cell, morula, and blastocyst stage embryos and isolate differentially expressed amplicons. Moreover, we have used a fluorescence monitored real time quantitative PCR to quantify and analyze the expression patterns of the target differentially expressed transcripts through out the preimplantation stages from oocytes to blastocyst. For this, total RNA isolated from bovine 8-cell (n,=,188), 16-cell (n,=,94), morula (n,=,35), and blastocyst (n,=,15) were reverse transcribed and subjected to DDRT-PCR. Target differentially expressed transcripts were quantified by real time quantitative PCR. The cDNA banding pattern analysis revealed that large number of cDNA bands were conserved at 8-cell and blastocyst stage with a slight decrease at the morula stage. A total of 16 amplicons were cloned and sequenced. All expressed sequence tags (ESTs), except 1C19, showed sequence similarity with known genes or ESTs in GenBank. Sixty-two percent (10/16) of cDNA bands representing differentially expressed genes originated from 8-cell stage and the rest derived from the 16-cell, morula, or blastocyst stage. The quantitative PCR analysis has validated the expression patterns of 75% (12/16) of our transcripts to be in agreement with the results of DDRT-PCR. However, the quantitative PCR results of four transcripts showed a deviation from the pattern seen in DDRT-PCR. In conclusion, we have successfully applied mRNA DDRT-PCR to identify and isolate stage-specific expressed genes in bovine preimplantation embryos. In addition to validating the results of DDRT-PCR, quantitative real time PCR provides quantitative data on the expression of target genes. Mol. Reprod. Dev. 66: 105,114, 2003. © 2003 Wiley-Liss, Inc. [source] | |||||||||||||