scholarly journals Quantifying spontaneous metastasis in a syngeneic mouse melanoma model using real time PCR

2017 ◽  
Author(s):  
Wentao Deng ◽  
Sarah L. McLaughlin ◽  
David J. Klinke
Keyword(s):  
Real Time ◽  
Tumor Cells ◽  
Real Time Pcr ◽  
Bioluminescence Imaging ◽  
Melanoma Metastasis ◽  
Spontaneous Metastasis ◽  
Syngeneic Mouse ◽  
Metastatic Cell ◽  
Metastatic Cells ◽  
Qpcr Method

Modeling metastasis in vivo with animals is a priority for both revealing mechanisms of tumor dissemination and developing therapeutic methods. While conventional intravenous injection of tumor cells provides an efficient and consistent system for studying tumor cell extravasation and colonization, studying spontaneous metastasis derived from orthotopic tumor sites has the advantage of modeling more aspects of the metastatic cascade, but is challenging as it is difficult to detect small numbers of metastatic cells. In this work, we developed an approach for quantifying spontaneous metastasis in the syngeneic mouse B16 system using real time PCR. We first transduced B16 cells with lentivirus expressing firefly luciferase Luc2 gene for bioluminescence imaging. Next, we developed a real time quantitative PCR (qPCR) method for the detection of luciferase-expressing, metastatic tumor cells in mouse lungs and other organs. To illustrate the approach, we quantified lung metastasis in both spontaneous and experimental scenarios using B16F0 and B16F10 cells in C57BL/6Ncrl and NOD-Scid Gamma (NSG) mice. We tracked B16 melanoma metastasis with both bioluminescence imaging and qPCR, which were found to be self-consistent. Using this assay, we can quantitatively detect one Luc2 positive tumor cells out of 104 tissue cells, which corresponds to a metastatic burden of 1.8x104 metastatic cells per whole mouse lung. More importantly, the qPCR method was at least a factor of 10 more sensitive in detecting metastatic cell dissemination and should be combined with bioluminescence imaging as a high-resolution, end-point method for final metastatic cell quantitation. Given the rapid growth of primary tumors in many mouse models, assays with improved sensitivity can provide better insight into biological mechanisms that underpin tumor metastasis.

scholarly journals Quantifying spontaneous metastasis in a syngeneic mouse melanoma model using real time PCR

The Analyst ◽  
2017 ◽  
Vol 142 (16) ◽  
pp. 2945-2953 ◽  
Author(s):  
Wentao Deng ◽  
Sarah L. McLaughlin ◽  
David J. Klinke
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Spontaneous Metastasis ◽  
Mouse Melanoma ◽  
Syngeneic Mouse ◽  
Tumor Dissemination ◽  
Mouse Melanoma Model ◽  
Melanoma Model ◽  
A Priority

Modeling metastasis in vivo with animals is a priority for both revealing mechanisms of tumor dissemination and developing therapeutic methods.

scholarly journals FusionCatcher - a tool for finding somatic fusion genes in paired-end RNA-sequencing data

2014 ◽  
Author(s):  
Daniel Nicorici ◽  
Mihaela Satalan ◽  
Henrik Edgren ◽  
Sara Kangaspeska ◽  
Astrid Murumagi ◽  
...  
Keyword(s):  
Real Time ◽  
Tumor Cells ◽  
Rna Sequencing ◽  
Real Time Pcr ◽  
Software Tool ◽  
Fusion Genes ◽  
Sequencing Data ◽  
Detection Rates ◽  
Somatic Fusion

FusionCatcher is a software tool for finding somatic fusion genes in paired-end RNA-sequencing data from human or other vertebrates. FusionCatcher achieves competitive detection rates and real-time PCR validation rates in RNA-sequencing data from tumor cells. FusionCatcher is available at http://code.google.com/p/fusioncatcher

BRAF V600E Mutation Appears Specific for Hairy Cell Leukemia Among Low and Intermediate Grade B-Cell Lymphomas: Utility of a Real Time PCR Based Approach for Detection

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2635-2635
Author(s):  
Mark Ewalt ◽  
Subhadra Nandula ◽  
Adrienne A. Phillips ◽  
Vundavalli Murty ◽  
Mahesh Mansukhani ◽  
...  
Keyword(s):  
Real Time ◽  
Tumor Cells ◽  
Real Time Pcr ◽  
Braf V600e Mutation ◽  
Braf V600e ◽  
Hairy Cell ◽  
Cell Leukemia ◽  
Pcr Assay ◽  
Neoplastic Cells ◽  
Intermediate Grade

Abstract Abstract 2635 BACKGROUND: Hairy cell leukemia (HCL) is a rare type of B-cell non Hodgkin lymphoma (B-NHL), which is characterized by neoplastic cells exhibiting slender cytoplasmic projections and an activated phenotype. Unlike other B-NHL, HCL lacks characteristic recurrent chromosome abnormalities and its etiology remains elusive. Recently, the BRAF V600E mutation was described to occur at a high frequency in HCL in contrast to other B-NHL, suggesting that the BRAF V600E mutation could represent a disease defining lesion and a possible therapeutic target. In this study, we determined the prevalence of the BRAF V600E mutation in a large series of low and intermediate grade B-NHL and assessed the relationship of this mutation, if any, with microsatellite instability (MSI). METHODS: DNA was extracted from various low and intermediate grade B-NHL (n=100), as defined by the 2008 World Health Organization Classification, which comprised HCL (n=10 from 6 patients), chronic lymphocytic leukemia/small lymphocytic lymphoma (n=22), mantle cell lymphoma (n=19), marginal zone lymphoma (n=22), follicular lymphoma (n=20) and lymphoplasmacytic lymphoma (n=7). Percentage of neoplastic cells was assessed by flow cytometry (n=99) or immunohistochemistry (n=1). Presence of BRAF V600E mutation was determined using a real time PCR assay using allele-specific hydrolysis (“Taqman”) probes. The detection limit of the assay was determined by diluting DNA extracted from a fresh HCL sample with normal high molecular weight DNA extracted by the same method. MSI analysis was performed using a multiplex reaction for 5 quasi monomorphic mononucleotide repeat markers (BAT-25, BAT-26, MONO-27, NR-21 and NR-24) and two highly polymorphic penta nucleotide markers (Penta C and Penta D) as sample identifiers. In the absence of normal DNA for comparison, a sample was considered MSI-H if greater than two markers showed an altered pattern, indeterminate if only two showed an alteration and MSS if no marker showed an altered allele. RESULTS: The ubiquitous occurrence and high specificity of the BRAF V600E mutation for HCL was confirmed in our series of B-NHL. The HCL samples consisted of 10 specimens from 6 patients and were the initial diagnostic specimen in 2 patients. The percentage of neoplastic cells in these samples ranged from 1% to 78.4% (median 18.2%). The BRAF V600E mutation was detected in 7 of 10 (70%) samples of HCL from 5 of 6 (83%) patients. The proportion of hairy cells was ≤5% in samples where the mutation was not detected. All 90 of the other low and intermediate grade B-NHL (>50% tumor burden) were negative for the V600E mutation. Compared to the reported sensitivity of approximately 30% tumor cells for detecting the BRAF V600E mutation by Sanger sequencing, our real time PCR assay allowed detection of the mutation in samples containing ≥9.8% tumor cells. Analysis of the HCL cases for MSI revealed that all cases had a microsatellite stable (MSS) phenotype. CONCLUSIONS: The BRAF V600E mutation appears specific for HCL among low and intermediate grade B-NHL and is not associated with microsatellite instability. These characteristics thus warrant inclusion in disease definition. Further refinements of a realtime PCR based approach for detecting the BRAF V600E mutation might be of utility in diagnosis and disease monitoring, as the neoplastic cellular yield is often limited in HCL due to underlying myelofibrosis. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Investigation on the deletion and duplication of PMP22 gene in patients with Charcot-Marie-Tooth using Real-time PCR in Chaharmahal and Bakhtiari and Isfahan Provinces

2019 ◽  
Vol 21 (6) ◽  
pp. 254-257
Author(s):  
Masoumeh Pourhadi ◽  
Morteza Hashemzadeh-Chaleshtori ◽  
Mostafa Gholami ◽  
Mohammad-Saeed Jami
Keyword(s):  
Gene Duplication ◽  
Real Time ◽  
Internal Control ◽  
Real Time Pcr ◽  
Pmp22 Gene ◽  
Charcot Marie Tooth ◽  
Qpcr Method ◽  
Pcr Method ◽  
Hereditary Patterns ◽  
Prevalent Form

Background and aims: Charcot-Marie-Tooth (CMT) is a common sensory-motor polyneuropathy with a prevalence of 1/2500. It is divided into different subgroups and has various hereditary patterns. Among the different subgroups of CMT, type 1A is the most prevalent form of the disease, which is created due to the duplication of the PMP22 gene. In patients has a deletion in the PMP22 gene, the hereditary neuropathic disease is known to be liable to pressure. The aim of this study was to identify the patients affected by the disease with the new, simple, and fast qPCR method and to investigate the appropriateness of this method in evaluating these types of mutations. Methods: In this analytical-descriptive study (code:IR.SKUMS.REC.1394.152), gene duplication and deletion in the patients were studied using the Excel software. The blood samples of 15 families afflicted with CMT and 49 healthy individuals were collected in EDTA anticoagulant tubes and analyzed. DNA extraction and quantitative real-time PCR method were applied for the PMP22 gene as the target gene and the albumin gene as the internal control gene. Results: Two genes were compared in each patient, and it was found that 46% of the subjects had duplication in the PMP22 gene. Conclusion: The qPCR method is an easy and fast way to detect gene duplication and deletion in CMT patients. It does not require any statistical software and can be performed without needing for parental DNA. In addition, the results of this study are consistent with the results of various studies in some countries of the world where the highest levels of deletion and duplication in PMP22 gene are seen.

scholarly journals Mycobacterium avium subsp. hominissuis infection in two sibling Fjord horses diagnosed using quantitative real time PCR: a case report

2011 ◽  
Vol 56 (No. 6) ◽  
pp. 294-301 ◽  
Author(s):  
M. Blahutkova ◽  
P. Fictum ◽  
M. Skoric ◽  
B. Bezdekova ◽  
P. Jahn ◽  
...  
Keyword(s):  
Real Time ◽  
Mycobacterium Avium ◽  
Real Time Pcr ◽  
Intestinal Content ◽  
Mycobacterium Avium Subsp ◽  
Pathological Findings ◽  
Quantitative Real Time Pcr ◽  
Mesenteric Lymph Nodes ◽  
Mesenteric Lymph ◽  
Qpcr Method

This report describes new possibilities for intravital and post mortem diagnosis of avian mycobacteriosis in horses using the quantitative real time PCR (qPCR) method. Using this method, Mycobacterium avium subsp. hominissuis was diagnosed in two sibling Fjord horses. In the first horse, M. a. hominissuis was detected by qPCR in numbers of 2.89 &times; 10<sup>5</sup> and 1.47 &times; 10<sup>4</sup> cells per 1 g of intestinal content and mesenteric lymph nodes, respectively; in the second horse, faeces and mesenteric lymph node samples showed numbers of 6.31 &times; 10<sup>5</sup> and 3.36 &times; 10<sup>6</sup> cells per 1 g of tissue, respectively. Another aim of this study was to comprehensively describe clinical and pathological findings in both animals.

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