scholarly journals Screening of miRNAs in plasma as a diagnostic biomarker for cardiac disease based on optimization of extraction and qRT-PCR condition assay through amplification efficiency

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Eunmi Ban ◽  
Haejin Kwon ◽  
Hong Seog Seo ◽  
Young Sook Yoo ◽  
Eun Joo Song
Keyword(s):  
Cardiac Disease ◽  
Annealing Temperature ◽  
Rna Extraction ◽  
Amplification Efficiency ◽  
Diagnostic Biomarker ◽  
Quantitative Real Time Pcr ◽  
Qrt Pcr ◽  
Ethanol Washing ◽  
Pcr Conditions ◽  
Plasma Mirna

Abstract Background Although quantitative real-time PCR (qRT-PCR) is a common and sensitive method for miRNAs analysis, it is necessary to optimize conditions and minimize qRT-PCR inhibitors to achieve reliable results. The aim of this study was to minimize interference by contaminants in qRT-PCR, maximize product yields for miRNA analyses, and optimize PCR conditions for the reliable screening of miRNAs in plasma. Methods The annealing temperature was first optimized by assessing amplification efficiencies. The effects of extraction conditions on levels of inhibitors that interfere with PCR were evaluated. The tested extraction conditions were the volume of the upper layer taken, number of chloroform extractions, and the inclusion of ethanol washing, a process that reduces PCR interference during RNA extraction using TRIzol. Results An acceptable amplification efficiency of RT-qPCR was achieved by the optimization of the annealing temperature of the tested miRNAs and by the collection a supernatant volume corresponding to about 50% of the volume of TRIzol with triple chloroform extraction. These optimal extraction and PCR conditions were successfully applied to plasma miRNA screening to detect biomarker candidates for the diagnosis of acute myocardial infarction. Conclusion This is the first study to optimize extraction and qRT-PCR conditions, while improving miRNA yields and minimizing the loss of extracted miRNA by evaluations of the amplification efficiency.

scholarly journals A probe-based qRT-PCR method to profile immunological gene expression in blood of captive beluga whales (Delphinapterus leucas)

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e3840 ◽  
Author(s):  
Ming-An Tsai ◽  
I-Hua Chen ◽  
Jiann-Hsiung Wang ◽  
Shih-Jen Chou ◽  
Tsung-Hsien Li ◽  
...  
Keyword(s):  
Gene Expression ◽  
Immune System ◽  
Reference Genes ◽  
Animal Health ◽  
Rna Extraction ◽  
Disease Onset ◽  
Delphinapterus Leucas ◽  
Amplification Efficiency ◽  
Beluga Whales ◽  
Qrt Pcr

Cytokines are fundamental for a functioning immune system, and thus potentially serve as important indicators of animal health. Quantitation of mRNA using quantitative reverse transcription polymerase chain reaction (qRT-PCR) is an established immunological technique. It is particularly suitable for detecting the expression of proteins against which monoclonal antibodies are not available. In this study, we developed a probe-based quantitative gene expression assay for immunological assessment of captive beluga whales (Delphinapterus leucas) that is one of the most common cetacean species on display in aquariums worldwide. Six immunologically relevant genes (IL-2Rα, -4, -10, -12, TNFα, and IFNγ) were selected for analysis, and two validated housekeeping genes (PGK1 and RPL4) with stable expression were used as reference genes. Sixteen blood samples were obtained from four animals with different health conditions and stored in RNAlater™ solution. These samples were used for RNA extraction followed by qRT-PCR analysis. Analysis of gene transcripts was performed by relative quantitation using the comparative Cq method with the integration of amplification efficiency and two reference genes. The expression levels of each gene in the samples from clinically healthy animals were normally distributed. Transcript outliers for IL-2Rα, IL-4, IL-12, TNFα, and IFNγ were noticed in four samples collected from two clinically unhealthy animals. This assay has the potential to identify immune system deviation from normal state, which is caused by health problems. Furthermore, knowing the immune status of captive cetaceans could help both trainers and veterinarians in implementing preventive approaches prior to disease onset.

Overexpression of Pygo2 Increases Differentiation of Human Umbilical Cord Mesenchymal Stem Cells into Cardiomyocyte-like Cells

2020 ◽  
Vol 20 (4) ◽  
pp. 318-324 ◽  
Author(s):  
Lei Yang ◽  
Shuoji Zhu ◽  
Yongqing Li ◽  
Jian Zhuang ◽  
Jimei Chen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Umbilical Cord ◽  
Heart Function ◽  
Critical Role ◽  
Human Umbilical Cord ◽  
Stem Cell Markers ◽  
Quantitative Real Time Pcr ◽  
Qrt Pcr

Background: Our previous studies have shown that Pygo (Pygopus) in Drosophila plays a critical role in adult heart function that is likely conserved in mammals. However, its role in the differentiation of human umbilical cord mesenchymal stem cells (hUC-MSCs) into cardiomyocytes remains unknown. Objective: To investigate the role of pygo2 in the differentiation of hUC-MSCs into cardiomyocytes. Methods: Third passage hUC-MSCs were divided into two groups: a p+ group infected with the GV492-pygo2 virus and a p− group infected with the GV492 virus. After infection and 3 or 21 days of incubation, Quantitative real-time PCR (qRT-PCR) was performed to detect pluripotency markers, including OCT-4 and SOX2. Nkx2.5, Gata-4 and cTnT were detected by immunofluorescence at 7, 14 and 21 days post-infection, respectively. Expression of cardiac-related genes—including Nkx2.5, Gata-4, TNNT2, MEF2c, ISL-1, FOXH1, KDR, αMHC and α-Actin—were analyzed by qRT-PCR following transfection with the virus at one, two and three weeks. Results : After three days of incubation, there were no significant changes in the expression of the pluripotency stem cell markers OCT-4 and SOX2 in the p+ group hUC-MSCs relative to controls (OCT-4: 1.03 ± 0.096 VS 1, P > 0.05, SOX2: 1.071 ± 0.189 VS 1, P > 0.05); however, after 21 days, significant decreases were observed (OCT-4: 0.164 ± 0.098 VS 1, P < 0.01, SOX2: 0.209 ± 0.109 VS 1, P < 0.001). Seven days following incubation, expression of mesoderm specialisation markers, such as Nkx2.5, Gata-4, MEF2c and KDR, were increased; at 14 days following incubation, expression of cardiac genes, such as Nkx2.5, Gata-4, TNNT2, MEF2c, ISL-1, FOXH1, KDR, αMHC and α-Actin, were significantly upregulated in the p+ group relative to the p− group (P < 0.05). Taken together, these findings suggest that overexpression of pygo2 results in more hUCMSCs gradually differentiating into cardiomyocyte-like cells. Conclusion: We are the first to show that overexpression of pygo2 significantly enhances the expression of cardiac-genic genes, including Nkx2.5 and Gata-4, and promotes the differentiation of hUC-MSCs into cardiomyocyte-like cells.

scholarly journals Development of an RNA Extraction Protocol for a Norovirus from Raw Oysters and Detection by qRT-PCR and Droplet-Digital RT-PCR

Foods ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1804
Author(s):  
Daniel Plante ◽  
Julio Alexander Bran Barrera ◽  
Maude Lord ◽  
Irène Iugovaz ◽  
Neda Nasheri
Keyword(s):  
Hepatitis A Virus ◽  
Rna Extraction ◽  
Complex Nature ◽  
Murine Norovirus ◽  
Rt Pcr ◽  
Pcr Inhibitors ◽  
Qrt Pcr ◽  
Extraction Protocol ◽  
Viral Particles ◽  
Hands On

Foodborne viruses such as norovirus and hepatitis A virus cause frequent outbreaks associated with the consumption of raw or undercooked oysters. Viral particles are bioaccumulated in the oyster’s digestive glands, making RNA extraction and RT-PCR detection difficult due to the complex nature of the food matrix and the presence of RT-PCR inhibitors. Herein, we have developed a viral RNA extraction protocol from raw oysters using murine norovirus (MNV) as a surrogate for human noroviruses. The method combines lysis in Tri-Reagent reagent, followed by RNA extraction using Direct-Zol purification columns and lithium chloride precipitation. Viral load quantification was performed by both qRT-PCR and droplet-digital RT-PCR. We have demonstrated that this method can efficiently remove RT-PCR inhibitors, and is sensitive enough to reliably detect viral contamination at 25 PFU/0.2 g. We have also compared the efficiency of this method with the ISO 15216-1:2017 method and Method E developed by Quang and colleagues, and observed significantly higher efficiency compared with the ISO 15216-1 method and comparable efficiency with Method E, with less steps, and shorter hands-on time.

scholarly journals Ascorbic acid improves parthenogenetic embryo development through TET proteins in mice

2019 ◽  
Vol 39 (1) ◽  
Author(s):  
Wei Gao ◽  
Xianfeng Yu ◽  
Jindong Hao ◽  
Ling Wang ◽  
Minghui Qi ◽  
...  
Keyword(s):  
Ascorbic Acid ◽  
Vitamin C ◽  
Embryonic Development ◽  
Real Time Pcr ◽  
Quantitative Real Time Pcr ◽  
Qrt Pcr ◽  
Tet Proteins ◽  
Parthenogenetic Embryo ◽  
Blastocyst Rate

Abstract The TET (Ten-Eleven Translocation) proteins catalyze the oxidation of 5mC (5-methylcytosine) to 5hmC (5-hydroxymethylcytosine) and play crucial roles in embryonic development. Ascorbic acid (Vc, Vitamin C) stimulates the expression of TET proteins, whereas DMOG (dimethyloxallyl glycine) inhibits TET expression. To investigate the role of TET1, TET2, and TET3 in PA (parthenogenetic) embryonic development, Vc and DMOG treatments were administered during early embryonic development. The results showed that Vc treatment increased the blastocyst rate (20.73 ± 0.46 compared with 26.57 ± 0.53%). By contrast, DMOG reduced the blastocyst rate (20.73 ± 0.46 compared with 11.18 ± 0.13%) in PA embryos. qRT-PCR (quantitative real-time PCR) and IF (immunofluorescence) staining results revealed that TET1, TET2, and TET3 expressions were significantly lower in PA embryos compared with normal fertilized (Con) embryos. Our results revealed that Vc stimulated the expression of TET proteins in PA embryos. However, treatment with DMOG significantly inhibited the expression of TET proteins. In addition, 5hmC was increased following treatment with Vc and suppressed by DMOG in PA embryos. Taken together, these results indicate that the expression of TET proteins plays crucial roles mediated by 5hmC in PA embryonic development.

Detection of COX-2 in liquid biopsy of patients with prostate cancer

2021 ◽  
pp. jclinpath-2021-207755
Author(s):  
Vanessa Silva Pereira ◽  
Beatriz da Costa Aguiar Alves ◽  
Jaques Waisberg ◽  
Fernando Fonseca ◽  
Flavia Gehrke
Keyword(s):  
Prostate Cancer ◽  
Slow Growth ◽  
Rna Extraction ◽  
Specific Antigen ◽  
Common Type ◽  
Control Group ◽  
Quantitative Real Time Pcr ◽  
Diagnosis And Prognosis ◽  
The Mean ◽  
Cox 2

AimsTo determine the profile of COX-2 gene expression in patients with prostate cancer attended at the ABC University Health Center outpatient clinic and correlate the results with patients’ anatomopathological examinations. Prostate cancer is the sixth most common type of cancer worldwide and the second in Brazil. COX-2 expression is associated with an unfavourable prognosis.Methods15.0 mL of peripheral blood were collected from 24 patients and 25 healthy men. RNA extraction was performed using the QIAamp RNA Blood Mini Kit. Complementary DNA synthesis was performed using SuperScript II RNAse Reverse Transcriptase. Quantitative real-time PCR was performed with specific COX-2 oligonucleotides and the endogenous GAPDH gene.ResultsThe mean age of the patients was 69 years old. The Gleason scoring system showed 37.5% of patients with Gleason 6 (slow growth, low risk), 45.8% with Gleason 7 (intermediate risk) and 16.7% with Gleason 8 or 9 (risk of high-grade cancer). The median COX-2 expression in the study group was 0.97, while in the control group it was 0.11 (p<0.045).ConclusionsPatients with prostate cancer showed higher COX-2 expression at diagnosis compared with the control group. Since COX-2 detection associated with prostate-specific antigen dosage shows promise as a biomarker for diagnosis and prognosis in patients with prostate cancer, further research is required to confirm these findings.

SOX9 stands for a bio-marker for prostate cancer detection

2020 ◽  
Author(s):  
Hao Zi ◽  
Wen-Lin Tao ◽  
Lei Gao ◽  
Zhao-Hua Yu ◽  
Xiao-Dong Bai ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Cancer Detection ◽  
Roc Curve ◽  
Operating Characteristic ◽  
Diagnostic Value ◽  
Quantitative Real Time Pcr ◽  
Optimal Cutoff ◽  
Qrt Pcr ◽  
Incidence And Mortality ◽  
The Relationship

Abstract Background Prostate cancer is one of common cancers around the world, and in our country the incidence and mortality of PCa are both increasing. More and more reports have revealed that SOX9 is involved in various human cancers. In this study, we aimed to explore the relationship between SOX9 expression and diagnostic value of PCa patients. Methods In this study, quantitative real-time PCR (qRT-PCR) was performed to determine the expression of SOX9 of the 131 PCa patients and 74 healthy volunteers. And receiver operating characteristic (ROC) curve was used to determine the diagnostic value of SOX9 for PCa patients. Results The results of qRT-PCR showed that the expression of serum SOX9 in PCa patients was higher than that in healthy controls (P < 0.05). And the expression of SOX9 was significantly associated with PSA (P = 0.001), differentiation (P = 0.000), and lymph node metastasis (P = 0.000). Besides, the area under the ROC curve (AUC) was 0.966 with the sensitivity of 93.2% and specificity of 87.8% respectively. The optimal cutoff value of SOX9 was 2.34. Conclusions Our results found that SOX9 is a novel oncogene for PCa, and may be a novel and effective biomarker for the diagnosis of patients with PCa.

scholarly journals Reference gene selection for quantitative real-time PCR (qRT-PCR) expression analysis in Galium aparine L.

PLoS ONE ◽  
2020 ◽  
Vol 15 (2) ◽  
pp. e0226668 ◽  
Author(s):  
Xu Su ◽  
Liuyang Lu ◽  
Yashe Li ◽  
Congai Zhen ◽  
Guilei Hu ◽  
...  
Keyword(s):  
Real Time ◽  
Reference Gene ◽  
Expression Analysis ◽  
Real Time Pcr ◽  
Gene Selection ◽  
Quantitative Real Time Pcr ◽  
Reference Gene Selection ◽  
Qrt Pcr ◽  
Galium Aparine ◽  
Selection For

scholarly journals A Simplified Quantitative Real-Time PCR Assay for Monitoring SARS-CoV-2 Growth in Cell Culture

mSphere ◽  
2020 ◽  
Vol 5 (5) ◽  
Author(s):  
Christian Shema Mugisha ◽  
Hung R. Vuong ◽  
Maritza Puray-Chavez ◽  
Adam L. Bailey ◽  
Julie M. Fox ◽  
...  
Keyword(s):  
Cell Culture ◽  
Real Time ◽  
Real Time Pcr ◽  
Rna Extraction ◽  
Quantitative Real Time Pcr ◽  
Pcr Assay ◽  
Dna Viruses ◽  
Culture Supernatants ◽  
Hiv 1 ◽  
Rna And Dna

ABSTRACT Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected millions within just a few months, causing severe respiratory disease and mortality. Assays to monitor SARS-CoV-2 growth in vitro depend on time-consuming and costly RNA extraction steps, hampering progress in basic research and drug development efforts. Here, we developed a simplified quantitative real-time PCR assay that bypasses viral RNA extraction steps and can monitor SARS-CoV-2 growth from a small amount of cell culture supernatants. In addition, we show that this approach is easily adaptable to numerous other RNA and DNA viruses. Using this assay, we screened the activities of a number of compounds that were predicted to alter SARS-CoV-2 entry and replication as well as HIV-1-specific drugs in a proof-of-concept study. We found that E64D (inhibitor of endosomal proteases cathepsin B and L) and apilimod (endosomal trafficking inhibitor) potently decreased the amount of SARS-CoV-2 RNA in cell culture supernatants with minimal cytotoxicity. Surprisingly, we found that the macropinocytosis inhibitor ethylisopropylamiloride (EIPA) similarly decreased SARS-CoV-2 RNA levels in supernatants, suggesting that entry may additionally be mediated by an alternative pathway. HIV-1-specific inhibitors nevirapine (a nonnucleoside reverse transcriptase inhibitor [NNRTI]), amprenavir (a protease inhibitor), and allosteric integrase inhibitor 2 (ALLINI-2) modestly inhibited SARS-CoV-2 replication, albeit the 50% inhibitory concentration (IC50) values were much higher than that required for HIV-1. Taking the data together, this simplified assay will expedite basic SARS-CoV-2 research, be amenable to mid-throughput screening assays (i.e., drug, CRISPR, small interfering RNA [siRNA], etc.), and be applicable to a broad number of RNA and DNA viruses. IMPORTANCE Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of the coronavirus disease 2019 (COVID-19) pandemic, is continuing to cause immense respiratory disease and social and economic disruptions. Conventional assays that monitor SARS-CoV-2 growth in cell culture rely on costly and time-consuming RNA extraction procedures, hampering progress in basic SARS-CoV-2 research and development of effective therapeutics. Here, we developed a simple quantitative real-time PCR assay to monitor SARS-CoV-2 growth in cell culture supernatants that does not necessitate RNA extraction and that is as accurate and sensitive as existing methods. In a proof-of-concept screen, we found that E64D, apilimod, EIPA, and remdesivir can substantially impede SARS-Cov-2 replication, providing novel insight into viral entry and replication mechanisms. In addition, we show that this approach is easily adaptable to numerous other RNA and DNA viruses. This simplified assay will undoubtedly expedite basic SARS-CoV-2 and virology research and be amenable to use in drug screening platforms to identify therapeutics against SARS-CoV-2.

scholarly journals A rapid RNA extraction method from oil palm tissues suitable for reverse transcription quantitative real-time PCR (RT-qPCR)

3 Biotech ◽  
2020 ◽  
Vol 10 (12) ◽  
Author(s):  
Siti Suriawati Badai ◽  
Omar Abd Rasid ◽  
Ghulam Kadir Ahmad Parveez ◽  
Mat Yunus Abdul Masani
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Oil Palm ◽  
Real Time Pcr ◽  
Extraction Method ◽  
Rna Extraction ◽  
Quantitative Real Time Pcr

Effect of anchor and core sequence in microsatellite primers on flax fingerprinting patterns

2001 ◽  
Vol 137 (1) ◽  
pp. 37-44 ◽  
Author(s):  
I. WIESNER ◽  
D. WIESNEROVÁ ◽  
E. TEJKLOVÁ
Keyword(s):  
Cluster Analysis ◽  
Large Scale ◽  
Annealing Temperature ◽  
Microsatellite Primers ◽  
Microsatellite Data ◽  
Core Group ◽  
The Core ◽  
Polymorphic Bands ◽  
Flax Cultivar ◽  
Pcr Conditions

The aim of this study was to select the best arrangements of MP–PCR (microsatellite-primed PCR) for routine large-scale fingerprinting of flax cultivars. We found optimum PCR conditions for the application of five previously published primers (PCT1–PCT5) to flax cultivar fingerprinting. We modified to optimum MP–PCR which was targeted to flax tetrameric [GATA] microsatellite loci specified by primer PCT6. We found that after a reamplification PCR step was involved we were able to generate highly discriminating fingerprinting patterns, which distinguished all eight flax cultivars individually. In particular primers 3PCT1 and 3PCT2 were promising for future large-scale fingerprinting due to the production of most polymorphic bands. Increasing annealing temperature within a temperature profile helped to generate new polymorphisms within flax microsatellite patterns especially with primer 3PCT2. Using this primer we succeeded in generating new polymorphic bands after increasing annealing temperature from 55 °C to 60 °C, and to 65 °C. A cluster analysis of flax cultivars was performed based on microsatellite data. The core group of eight flax cultivars was clustered into two homogeneous subclusters. A lower level of cultivar clustering within subclusters was not detected.

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