scholarly journals Up-regulated miR-500a enhances hepatocarcinoma metastasis by repressing PTEN expression

2017 ◽  
Vol 37 (6) ◽  
Author(s):  
Yufeng Zhao ◽  
Yuehui Wang ◽  
Yaqiang Wang
Keyword(s):  
Protein Expression ◽  
Molecular Mechanism ◽  
Reverse Transcription ◽  
Qrt Pcr ◽  
Reverse Transcription Pcr ◽  
Normal People ◽  
Hepatocarcinoma Cell ◽  
Proliferation And Metastasis ◽  
Phosphatase And Tensin Homologue ◽  
The Relationship

It has been shown that miR-500a may play an important role in the metastasis of hepatocarcinoma. The present study is to explore the influence of miR-500a on hepatocarcinoma proliferation and metastasis, and the related molecular mechanism. The levels of miR-500a in the serum and tissues of patients with metastatic or non-metastatic hepatocarcinoma or normal people were determined by quantitative reverse transcription-PCR (qRT-PCR). The proliferation, invasion, and cloning of hepatocarcinoma cell lines SMMC-7721 after transfection with mimic miR-500a or inhibitor miR-500a were determined. Luciferase reported assay was used to explore the relationship between miR-500a and phosphatase and tensin homologue (PTEN). Then, the protein expression of PTEN, p-Akt (S473), p-Akt (T308), Akt, p-mTOR, mTOR, p-4E-BP1, 4E-BP1, p-S6K, and S6K in SMMC-7721 cells were also determined by Western blotting. The expression of miR-500a in patients with metastatic hepatocarcinoma was significantly higher than the non-metastatic hepatocarcinoma. Overexpression of miR-500a promoted the proliferation, invasion, and cloning of SMMC-7721 cells. Luciferase reported assay showed miR-500a could directly target at 3′-UTR of PTEN. Overexpression of miR-500a significantly reduced the expression of PTEN, and enhanced phosphorylation of Akt, mTOR, S6K, and 4E-BP1. In conclusion, the expression of miR-500a was related to the proliferation and metastasis of hepatocarcinoma, which may be partly because of the activation of AKT/mTOR pathway through targetting PTEN.

MiR-493 Induces Cytotoxic Autophagy in Prostate Cancer Cells through Regulation on PHLPP2

2020 ◽  
Vol 21 (14) ◽  
pp. 1451-1456 ◽  
Author(s):  
Jun Deng ◽  
Ming Ma ◽  
Wei Jiang ◽  
Liangliang Zheng ◽  
Suping Cui
Keyword(s):  
Prostate Cancer ◽  
Protein Expression ◽  
Cell Function ◽  
Mrna Levels ◽  
Prostate Cancer Cells ◽  
Potent Inducer ◽  
Qrt Pcr ◽  
Autophagy Gene ◽  
The Relationship ◽  
Cancer Inhibition

Background: MiR-493 promotes the proliferation of prostate cancer (PC) cells by targeting PHLPP2. We aimed to explore the relationship between miR-493 and autophagy in PC. Methods: qRT-PCR and western blotting were used to determine the mRNA levels and protein expression of miR-493, PHLPP2, autophagy gene BECN1 and ATG7 in PC cells. The autophagy gene expression was determined after PC cells transfected with miR-493 precursor or PHLPP2 precursor. Corresponding changes of autophagy phenotype and PC cell function were also studied. Results: The mRNA levels and protein expression of miR-493, PHLPP2, BECN1 and ATG7 in PC cells were significantly decreased in PC cells. Overexpression of miR-493 or PHLPP2 markedly upregulated the expression levels of BECN1 and ATG7 in PC cells. Overexpression of miR-493 and PHLPP2 markedly promoted autophagy, and inhibited the invasion and cloning formation of PC cells. Conclusion: MiR-493 is a potent inducer of cytotoxic autophagy that leads to prostate cancer inhibition by regulating on PHLPP2.

An Eco-friendly Fabrication of Silver Chloride Nanoparticles (AgCLNPs) using Onopordum acanthium L extract Induces Apoptosis in Breast Cancer MDA-MB-232 Cells

2021 ◽  
Author(s):  
Seyedeh Negin Shahcheraghi ◽  
Seyed Ataollah Sadat Shandiz ◽  
Bahareh Pakpour
Keyword(s):  
Breast Cancer ◽  
Real Time ◽  
Cell Lines ◽  
Reverse Transcription ◽  
Hek293 Cell ◽  
Silver Chloride ◽  
Hek293 Cells ◽  
Qrt Pcr ◽  
Reverse Transcription Pcr ◽  
Onopordum Acanthium

Abstract In the current experimental work, silver chloride nanoparticles (AgClNPs) were fabricated using Onopordum acanthium L extract and their apoptotic and cytotoxicity properties on breast cancer MDA_MB232 and normal HEK293 cell lines were also evaluated. AgClNPs formation was determined by X-ray diffraction (XRD), scanning electron microscopy (SEM), and energy dispersive spectroscopy (EDS) profile. Effect of fabricated AgClNPs on MDA_MB232 and HEK293 cells viability was performed using colorimetric MTT assay. Alterations in the mRNA expression levels of CAD and Bax genes in MDA-MB-232 cells were done using quantitative real-time reverse transcription-PCR (qRT-PCR) method. Subsequently, apoptotic properties were determined using flow cytometry and fluorescence microscopy studies. MTT results investigated that AgCLNPs have a significant dose-dependent lethal activity on MDA_MB232 compared to HEK293 cell lines. Quantitative real-time reverse transcription-PCR (qRT-PCR) results have also shown that AgCLNPs could up-regulate the apoptotic Bax and CAD gene expressions in the MDA_MB232 cells. Additionally, apoptotic assessment was performed by cell cycle analysis, annexin V/PI test, Hoescht 33258 dye, acridine orange and ethidium bromide (AO/EB) staining along with the detection of the reactive oxygen species (ROS) generation. Our results suggest that novel silver chloride nanoparticles fabricated by Onopordum acanthium L extract can display some promising cytotoxic properties through inducing apoptosis pathway.

scholarly journals Detection of mRNA by Reverse Transcription-PCR as an Indicator of Viability in Escherichia coliCells

1998 ◽  
Vol 64 (4) ◽  
pp. 1313-1318 ◽  
Author(s):  
G. E. C. Sheridan ◽  
C. I. Masters ◽  
J. A. Shallcross ◽  
B. M. Mackey
Keyword(s):  
Reverse Transcription ◽  
Room Temperature ◽  
Cellular Viability ◽  
Ethanol Treatment ◽  
Rt Pcr ◽  
Promising Candidate ◽  
Mrna Targets ◽  
Reverse Transcription Pcr ◽  
Different Types ◽  
The Relationship

ABSTRACT The relationship between the detection of mRNA and cellular viability in Escherichia coli was investigated in cells killed by heat or ethanol. Reverse transcription-PCR (RT-PCR) methods were developed for detecting mRNA from rpoH,groEL, and tufA genes. mRNA from all three genes was detected immediately after the cells had been killed by heat or ethanol but gradually disappeared with time when dead cells were held at room temperature. In heat-killed cells, some mRNA targets became undetectable after 2 to 16 h, whereas after ethanol treatment, mRNA was still detected after 16 h. In contrast, 16S rRNA was detected by RT-PCR in all samples containing dead cells and did not disappear during a subsequent incubation of 16 h at room temperature. Of the different types of nucleic acid, mRNA is the most promising candidate for an indicator of viability in bacteria, but its persistence in dead cells depends on the inactivating treatment and subsequent holding conditions.

scholarly journals NewIn SituCapture Quantitative (Real-Time) Reverse Transcription-PCR Method as an Alternative Approach for Determining Inactivation of Tulane Virus

2014 ◽  
Vol 80 (7) ◽  
pp. 2120-2124 ◽  
Author(s):  
Dapeng Wang ◽  
Shuxia Xu ◽  
David Yang ◽  
Glenn M. Young ◽  
Peng Tian
Keyword(s):  
Tissue Culture ◽  
Reverse Transcription ◽  
Viral Genome ◽  
Viral Infectivity ◽  
Qrt Pcr ◽  
Reverse Transcription Pcr ◽  
Alternative Approach ◽  
Tulane Virus ◽  
Pcr Method

ABSTRACTHuman noroviruses (HuNoVs) are the major cause of epidemic nonbacterial gastroenteritis. Although quantitative (real-time) reverse transcription-PCR (qRT-PCR) is widely used for detecting HuNoVs, it only detects the presence of viral RNA and does not indicate viral infectivity. Human blood group antigens (HBGAs) have been identified as receptors/co-receptors for both HuNoVs and Tulane virus (TV) and are crucial for viral infection. We propose that viral infectivity can be evaluated with a molecular assay based on receptor-captured viruses. In this study, we employed TV as an HuNoV surrogate to validate the HBGA-based capture qRT-PCR method against the 50% tissue culture infectious dose (TCID50) method. We employed type B HBGA on an immuno-well module to concentrate TV, followed by amplification of the captured viral genome byin situqRT-PCR. We first demonstrated that thisin situcapture qRT-PCR (ISC-qRT-PCR) method could effectively concentrate and detect TV. We then treated TV under either partial or full inactivation conditions and measured the remaining infectivity by ISC-qRT-PCR and a tissue culture-based amplification method (TCID50). We found that the ISC-qRT-PCR method could be used to evaluate virus inactivation deriving from damage to the capsid and study interactions between the capsid and viral receptor. Heat, chlorine, and ethanol treatment primarily affect the capsid structure, which in turns affects the ability of the capsid to bind to viral receptors. Inactivation of the virus by these methods could be reflected by the ISC-qRT-PCR method and confirmed by TCID50assay. However, the loss of the infectivity caused by damage to the viral genome (such as that from UV irradiation) could not be effectively reflected by this method. Despite this limitation, the ISC-qRT-PCR provides an alternative approach to determine inactivation of Tulane virus. A particular advantage of the ISC-qRT-PCR method is that it is also a faster and easier method to effectively recover and detect the viruses, as there is no need to extract viral RNA or to transfer the captured virus from magnetic beads to PCR tubes for further amplification. Therefore, ISC-qRT-PCR can be easily adapted for use in automated systems for multiple samples.

scholarly journals Real-time reverse transcription PCR (qRT-PCR) and its potential use in clinical diagnosis

2005 ◽  
Vol 109 (4) ◽  
pp. 365-379 ◽  
Author(s):  
Stephen A. Bustin ◽  
Reinhold Mueller
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Therapy Monitoring ◽  
Clinical Diagnostics ◽  
Clinical Diagnostic Laboratory ◽  
Diagnostic Laboratory ◽  
Qrt Pcr ◽  
Reverse Transcription Pcr ◽  
Clinical Diagnostic ◽  
Pcr Assays

qRT-PCR (real-time reverse transcription-PCR) has become the benchmark for the detection and quantification of RNA targets and is being utilized increasingly in novel clinical diagnostic assays. Quantitative results obtained by this technology are not only more informative than qualitative data, but simplify assay standardization and quality management. qRT-PCR assays are most established for the detection of viral load and therapy monitoring, and the development of SARS (severe acute respiratory syndrome)-associated coronavirus qRT-PCR assays provide a textbook example of the value of this technology for clinical diagnostics. The widespread use of qRT-PCR assays for diagnosis and the detection of disease-specific prognostic markers in leukaemia patients provide further examples of their usefulness. Their value for the detection of disease-associated mRNA expressed by circulating tumour cells in patients with solid malignancies is far less apparent, and the clinical significance of results obtained from such tests remains unclear. This is because of conceptual reservations as well as technical limitations that can interfere with the diagnostic specificity of qRT-PCR assays. Therefore, although it is evident that qRT-PCR assay has become a useful and important technology in the clinical diagnostic laboratory, it must be used appropriately and it is essential to be aware of its limitations if it is to fulfil its potential.

scholarly journals Comparison of Automated Quantitative Reverse Transcription-PCR and Direct Fluorescent-Antibody Detection for Routine Rabies Diagnosis in the United States

2015 ◽  
Vol 53 (9) ◽  
pp. 2983-2989 ◽  
Author(s):  
Michelle Dupuis ◽  
Scott Brunt ◽  
Kim Appler ◽  
April Davis ◽  
Robert Rudd
Keyword(s):  
United States ◽  
Reverse Transcription ◽  
Gold Standard ◽  
Fluorescent Antibody ◽  
The United States ◽  
Pcr Assay ◽  
Qrt Pcr ◽  
Reverse Transcription Pcr ◽  
Direct Fluorescent Antibody ◽  
Rabies Diagnosis

Rabies virus found worldwide and prevalent throughout the United States continues to be a public health concern. Direct-fluorescent antibody (DFA) detection remains the gold standard for rabies virus diagnostics. Assessing the utility of a high-throughput molecular platform such as the QIAsymphony SP/AS, in conjunction with quantitative reverse transcription-PCR (qRT-PCR), to augment or potentially replace the DFA test, was the focus of this project. Here we describe a triplex qRT-PCR assay, including assembly and evaluation for sensitivity, specificity, and ability to detect variants. Additionally, we compared the qRT-PCR assay to the gold standard direct fluorescent-antibody test. More than 1,000 specimens submitted for routine rabies diagnosis were tested to directly compare the two methods. All results were in agreement between the two methods, with one additional specimen detected by qRT-PCR below the limits of the DFA sensitivity. With the proper continued validation for variant detection, molecular methods have a place in routine rabies diagnostics within the United States.

scholarly journals Rapid Detection of Novel Coronavirus (COVID-19) by Reverse Transcription-Loop-Mediated Isothermal Amplification

2020 ◽  
Author(s):  
Laura E. Lamb ◽  
Sarah N. Bartolone ◽  
Elijah Ward ◽  
Michael B. Chancellor
Keyword(s):  
Diagnostic Test ◽  
Reverse Transcription ◽  
Simulated Patient ◽  
Isothermal Amplification ◽  
Rapid Screening ◽  
Health Concern ◽  
Loop Mediated Isothermal Amplification ◽  
Qrt Pcr ◽  
Reverse Transcription Pcr ◽  
Novel Coronavirus

AbstractNovel Corona virus (COVID-19 or 2019-nCoV) is an emerging global health concern that requires a rapid diagnostic test. Quantitative reverse transcription PCR (qRT-PCR) is currently the standard for COVID-19 detection; however, Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) may allow for faster and cheaper field based testing at point-of-risk. The objective of this study was to develop a rapid screening diagnostic test that could be completed in under 30 minutes. Simulated patient samples were generated by spiking serum, urine, saliva, oropharyngeal swabs, and nasopharyngeal swabs with a portion of the COVID-19 nucleic sequence. The samples were tested using RT-LAMP as well as by conventional qRT-PCR. Specificity of the RT-LAMP was evaluated by also testing against other related coronaviruses. RT-LAMP specifically detected COVID-19 in simulated patient samples. This test was performed in under 30 minutes. This approach could be used for monitoring of exposed individuals or potentially aid with screening efforts in the field and potential ports of entry.

scholarly journals Comparison of three TaqMan Real-Time Reverse Transcription-PCR assays in detecting SARS-CoV-2

2020 ◽  
Author(s):  
Yan Xiao ◽  
Zhen Li ◽  
Xinming Wang ◽  
Yingying Wang ◽  
Ying Wang ◽  
...  
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Detection Rate ◽  
N Gene ◽  
Detection Rates ◽  
Qrt Pcr ◽  
Reverse Transcription Pcr ◽  
Linear Detection ◽  
Gene Assay ◽  
Pcr Assays

AbstractQuick and accurate detection of SARS-CoV-2 is critical for COVID-19 control. Dozens of real-time reverse transcription PCR (qRT-PCR) assays have been developed to meet the urgent need of COVID-19 control. However, methodological comparisons among the developed qRT-PCR assays are limited. In the present study, we evaluated the sensitivity, specificity, amplification efficiency, and linear detection ranges of three qRT-PCR assays, including the assays developed by our group (IPBCAMS), and the assays recommended by WHO and China CDC (CCDC). The three qRT-PCR assays exhibited similar sensitivities, with the limit of detection (LOD) at about 10 copies per reaction (except the ORF 1b gene assay in CCDC assays with a LOD at about 100 copies per reaction). No cross reaction with other respiratory viruses were observed in all of the three qRT-PCR assays. Wide linear detection ranges from 106 to 101 copies per reaction and acceptable reproducibility were obtained. By using 25 clinical specimens, the N gene assay of IPBCAMS assays and CCDC assays performed better (with detection rates of 92% and 100%, respectively) than that of the WHO assays (with a detection rate of 60%), and the ORF 1b gene assay in IPBCAMS assays performed better (with a detection rate of 64%) than those of the WHO assays and the CCDC assays (with detection rates of 48% and 20%, respectively). In conclusion, the N gene assays of CCDC assays and IPBCAMS assays and the ORF 1b gene assay of IPBCAMS assays were recommended for qRT-PCR screening of SARS-CoV-2.

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