scholarly journals Introducing Novel Molecular-based Method for Quantification of Homologous Recombination Efficiency

2021 ◽  
Author(s):  
Mustapha Dibbasey ◽  
Terry Gaymes
Keyword(s):  
Homologous Recombination ◽  
Real Time ◽  
Cell Lines ◽  
Real Time Pcr ◽  
Cost Effective ◽  
Lacz Gene ◽  
Expression Arrays ◽  
Recombinant Product ◽  
Wide Range ◽  
High Level

AbstractBackgroundHomologous recombination (HR) pathway is a DNA double-stranded breaks repair pathway well-known for its high level of accuracy. Low HR pathway efficiency clinically known as homologous recombination deficiency (HRD) was identified in some cancers such as breast and ovarian cancers and studies have reported the sensitivity of HRD cancer cells to DNA repair inhibitors such as Olaparib. However, current techniques including immunofluorescence-based technique are qualitative-based, hence lack sensitivity to determine the functionality of HR pathway. Additionally, some of the techniques including gene expression arrays require expression study of wide range genes involve in HR pathway, which is not cost-effective. The aim of the study is to optimise a PCR-based assay (Norgen’s Homologous Recombination kit) that can be employed to quantitate HR efficiency in cells, which accurately reflects the functional status of HR pathway.Methods and FindingsThe kit has two test plasmids (dl-1 and dl-2) with partial deletions in the LacZ gene and the plasmids are generated from modification of pUC19. HR-proficient (HeLa and AsPC-1) and HR-deficient (CAPAN-1 cells) cancer cell lines were transfected with the two plasmids to generate functional LacZ gene (i.e. recombinant product). The recombinant product was quantified by real-time PCR. Although recombinant product was generated in all the cell lines, our real-time PCR demonstrated a high quantity of recombinant product in HeLa cell line whilst low quantity in CAPAN-1 and AsPC-1 cell lines. The quantity of recombinant product generated and quantified reflects HR pathway efficiency.ConclusionOverall, the results have provided some evidence that the PCR-based kit can be suitably employed for quantification of HR efficiency provided appropriate transfection method and reagent are used. However, further study is required to confirm HR efficiency status of AsPC-1 cells to ascertain the low HR efficiency detected by the kit in these cells.

scholarly journals Development of cost-effective real-time PCR test: to detect a wide range of HBV DNA concentrations in the western amazon region of Brazil

2014 ◽  
Vol 11 (1) ◽  
Author(s):  
Alcione de Oliveira dos Santos ◽  
Luan Felipo Botelho Souza ◽  
Lourdes Maria Borzacov ◽  
Juan Miguel Villalobos-Salcedo ◽  
Deusilene Souza Vieira
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Cost Effective ◽  
Hbv Dna ◽  
Amazon Region ◽  
Wide Range ◽  
Pcr Test

scholarly journals Development of a sensitivity and cost-effective real time pcr: measuring a wide range of HBV DNA concentrations

2013 ◽  
Author(s):  
Alcione Santos ◽  
Luan Souza ◽  
Lourdes Borzacov ◽  
Juan Villalobos-Salcedo ◽  
Deusilene Vieira
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Cost Effective ◽  
Hbv Dna ◽  
Wide Range

scholarly journals In-house reverse transcriptase polymerase chain reaction for detection of SARS-CoV-2 with increased sensitivity

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Manash Jyoti Kalita ◽  
Kalpajit Dutta ◽  
Gautam Hazarika ◽  
Ridip Dutta ◽  
Simanta Kalita ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Real Time ◽  
Real Time Pcr ◽  
Cost Effective ◽  
Sybr Green ◽  
Green Approach ◽  
Novel Approach ◽  
Effective Manner ◽  
Wide Range ◽  
Thermal Cycler

AbstractAs the COVID-19 infection continues to ravage the world, the advent of an efficient as well as the economization of the existing RT-PCR based detection assay essentially can become a blessing in these testing times and significantly help in the management of the pandemic. This study demonstrated an innovative and rapid corroboration of COVID-19 test based on innovative multiplex PCR. An assessment of optimal PCR conditions to simultaneously amplify the SARS-CoV-2 genes E, S and RdRp has been made by fast-conventional and HRM coupled multiplex real-time PCR using the same sets of primers. All variables of practical value were studied by amplifying known target-sequences from ten-fold dilutions of archived positive samples of COVID-19 disease. The multiplexing with newly designed E, S and RdRp primers have shown an efficient amplification of the target region of SARS-CoV-2. A distinct amplification was observed in 37 min using thermal cycler while it took 96 min in HRM coupled real time detection using SYBR green over a wide range of template concentrations. Our findings revealed decent concordance with other commercially available detection kits. This fast HRM coupled multiplex real-time PCR with SYBR green approach offers rapid and sensitive detection of SARS-CoV-2 in a cost-effective manner apart from the added advantage of primer compatibility for use in conventional multiplex PCR. The highly reproducible novel approach can propel extended applicability for developing sustainable commercial product besides providing relief to a resource limited setting.

scholarly journals Cost-Effective Real-Time Metabolic Profiling of Cancer Cell Lines for Plate-Based Assays

Chemosensors ◽  
2021 ◽  
Vol 9 (6) ◽  
pp. 139
Author(s):  
Wiktoria Blaszczak ◽  
Zhengchu Tan ◽  
Pawel Swietach
Keyword(s):  
Real Time ◽  
Cell Lines ◽  
Acid Production ◽  
Metabolic Regulation ◽  
Cost Effective ◽  
Culture Conditions ◽  
Metabolic Inhibitors ◽  
Metabolic Phenotype ◽  
Ductal Adenocarcinoma ◽  
Oxygen Utilization

A fundamental phenotype of cancer cells is their metabolic profile, which is routinely described in terms of glycolytic and respiratory rates. Various devices and protocols have been designed to quantify glycolysis and respiration from the rates of acid production and oxygen utilization, respectively, but many of these approaches have limitations, including concerns about their cost-ineffectiveness, inadequate normalization procedures, or short probing time-frames. As a result, many methods for measuring metabolism are incompatible with cell culture conditions, particularly in the context of high-throughput applications. Here, we present a simple plate-based approach for real-time measurements of acid production and oxygen depletion under typical culture conditions that enable metabolic monitoring for extended periods of time. Using this approach, it is possible to calculate metabolic fluxes and, uniquely, describe the system at steady-state. By controlling the conditions with respect to pH buffering, O2 diffusion, medium volume, and cell numbers, our workflow can accurately describe the metabolic phenotype of cells in terms of molar fluxes. This direct measure of glycolysis and respiration is conducive for between-runs and even between-laboratory comparisons. To illustrate the utility of this approach, we characterize the phenotype of pancreatic ductal adenocarcinoma cell lines and measure their response to a switch of metabolic substrate and the presence of metabolic inhibitors. In summary, the method can deliver a robust appraisal of metabolism in cell lines, with applications in drug screening and in quantitative studies of metabolic regulation.

scholarly journals The FMS like Tyrosine Kinase 3 (FLT3) Is Overexpressed in a Subgroup of Multiple Myeloma Patients with Inferior Prognosis

Cancers ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 2341
Author(s):  
Normann Steiner ◽  
Karin Jöhrer ◽  
Selina Plewan ◽  
Andrea Brunner-Véber ◽  
Georg Göbel ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Tyrosine Kinase ◽  
Real Time ◽  
Cell Lines ◽  
Real Time Pcr ◽  
Tyrosine Kinase Receptor ◽  
Bone Marrow Biopsies ◽  
Flt3 Inhibitors ◽  
Flt3 Expression

Therapy resistance remains a major challenge in the management of multiple myeloma (MM). We evaluated the expression of FLT3 tyrosine kinase receptor (FLT3, CD135) in myeloma cells as a possible clonal driver. FLT3 expression was analyzed in bone marrow biopsies of patients with monoclonal gammopathy of undetermined significance or smoldering myeloma (MGUS, SMM), newly diagnosed MM (NDMM), and relapsed/refractory MM (RRMM) by immunohistochemistry (IHC). FLT3 gene expression was analyzed by RNA sequencing (RNAseq) and real-time PCR (rt-PCR). Anti-myeloma activity of FLT3 inhibitors (midostaurin, gilteritinib) was tested in vitro on MM cell lines and primary MM cells by 3H-tymidine incorporation assays or flow cytometry. Semi-quantitative expression analysis applying a staining score (FLT3 expression IHC-score, FES, range 1–6) revealed that a high FES (>3) was associated with a significantly shorter progression-free survival (PFS) in NDMM and RRMM patients (p = 0.04). RNAseq and real-time PCR confirmed the expression of FLT3 in CD138-purified MM samples. The functional relevance of FLT3 expression was corroborated by demonstrating the in vitro anti-myeloma activity of FLT3 inhibitors on FLT3-positive MM cell lines and primary MM cells. FLT3 inhibitors might offer a new targeted therapy approach in a subgroup of MM patients displaying aberrant FLT3 signaling.

scholarly journals Increased Expression of the Pro-Protein Convertase Furin Predicts Decreased Survival in Ovarian Cancer

2007 ◽  
Vol 29 (4) ◽  
pp. 289-299
Author(s):  
Robert E. Page ◽  
Andrés J. P. Klein-Szanto ◽  
Samuel Litwin ◽  
Emmanuelle Nicolas ◽  
Raid Al-Jumaily ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Tumor Progression ◽  
Real Time ◽  
Cell Lines ◽  
Cancer Cell ◽  
Real Time Pcr ◽  
Ovarian Tumor ◽  
Cancer Cell Lines ◽  
Rt Pcr ◽  
Primary Tumors

Background: Proprotein convertases (PCs) are serine proteases that after restricted proteolysis activate many proteins that play a crucial role in cancer such as metalloproteinases, growth factors and growth factor receptors, adhesion molecules, and angiogenic factors. Although the expression of several PCs is increased in many tumors, their expression in primary ovarian tumors has not been studied in detail. We sought to determine if there was an association between the expression of the ubiquitously expressed PCs, furin, PACE-4, PC-5 and PC-7, and ovarian tumor progression. Methods: We assessed their expression by RT-PCR, Real-time PCR, Western blot, and immunohistochemistry using cells derived from normal human ovarian surface epithelium (HOSE) and cancer cell lines as well as ovarian epithelial cancer specimens (45 RT-PCR/Real-time PCR, and 120 archival specimens for Immunohistochemistry). Results: We found that furin expression was restricted to the cancer cell lines. In contrast, PACE-4 and PC-7 showed expression only in normal HOSE cells lines. Furthermore, furin was predominantly expressed in primary tumors from patients who survived for less than five years. The other PCs are either expressed in the group of survivors (PC-7 and PACE4) or expressed in low amounts (PC-5). Conclusions: Our studies point to a clear relationship between furin and ovarian cancer. In addition, these results show that furin exhibits the closest association with ovarian cancer among the ubiquitously expressed PCs, arguing against the redundancy of these proteases. In summary, furin may constitute a marker for ovarian tumor progression and could contribute to predict the outcome of this disease.

385 MIR-24-3P REGULATES CDX2 DURING THE PROCESS OF INTESTINALIZATION OF THE CARDIAC-TYPE EPITHELIUM IN A HUMAN MODEL OF BARRETT’S ESOPHAGUS

2021 ◽  
Vol 34 (Supplement_1) ◽  
Author(s):  
Manuel Pera ◽  
Marta Garrido ◽  
Gabriel Gil ◽  
Matteo Fassan ◽  
Marta Climent ◽  
...  
Keyword(s):  
Real Time ◽  
Barrett’S Esophagus ◽  
Esophageal Adenocarcinoma ◽  
Cell Lines ◽  
Real Time Pcr ◽  
Barrett's Esophagus ◽  
Intermediate Stage ◽  
Human Model ◽  
Adenocarcinoma Cell ◽  
Cdx2 Expression

Abstract   Cardiac-type epithelium has been proposed as an intermediate stage between normal squamous epithelium and intestinal metaplasia in the development of Barrett’s esophagus. Deregulation of certain miRNAs and their effects on CDX2 expression might contribute to the intestinalization process of cardiac-type epithelium. The aim of this study was to identify miRNAs differentially expressed between CDX2 positive and negative glands of Barrett’s esophagus and to examine the function of specific miRNAs on the regulation of CDX2. Methods miRNA expression profiling using OpenArrayTM analysis in microdissected cardiac-type glands with and without fully CDX2 expression was performed in biopsies from patients who developed cardiac-type epithelium in the remnant esophagus after esophagectomy. Data were validated using real-time PCR in esophageal adenocarcinoma cell lines and in situ and real-time PCR miRNA/CDX2/MUC2 co-expression analysis in cardiac-type mucosa samples. The effect of miR-24-3p precursor transfection on CDX2 expression was assessed in the esophageal adenocarcinoma cell lines FLO-1 and KYAE-1. Results CDX2 positive glands were characterized by an unique miRNA profile with a significant downregulation of miR-24-3p, miR-520e-3p, miR-548a-1, miR-597-5p, miR-133a-3p, miR-30a-5p, miR-638, miR-625-3p, miR-1255b-1, miR-1260a and upregulation of miR-590 (Figure 1A). miRNA-24-3p was identified as potential regulator of CDX2 gene expression in three bioinformatics algorithms, and this was confirmed in esophageal adenocarcinoma cell lines (Figure 1C). Furthermore, miR-24-3p expression negatively correlates with CDX2 in cardiac-type mucosa samples with different stages of mucosal intestinalization (Figure 1B). Conclusion These results imply that miRNA-24-3p directly targets CDX2, and downregulation of miRNA-24-3p is associated with the acquisition of an intestinal phenotype in cardiac-type epithelium.

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