quantitative polymerase chain reaction
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2022 ◽  
Vol 43 (2) ◽  
pp. 889-894
Nelson Jessé Rodrigues dos Santos ◽  
Renan Bressianini do Amaral ◽  
Luiz Ricardo Gonçalves ◽  
Rogério Ribas Lange ◽  

Bartonella is an emerging group of facultative intracellular bacteria causing circulatory and systemic disorders. Hosts for Bartonella are mostly mammals, specifically rodents, having a growing number of Bartonella species related to their infection. Capybaras (Hydrochoerus hydrochaeris) are abundant native rodents of Brazil, commonly found in urban parks. In the present study, we aimed to perform molecular screening of capybaras for Bartonella spp. Blood samples were collected from 17 free-ranging animals captured in Paraná State, Southern Brazil. None of the collected samples tested positive for the Bartonella-nuoG gene by quantitative polymerase chain reaction (qPCR), although all of them successfully amplified the mammal endogenous glyceraldehyde-3-phosphate dehydrogenase (gapdh) gene. Additionally, all animals were infested exclusively by Amblyomma dubitatum ticks at the time of sampling. This study was part of an active surveillance program, which is critical for monitoring animal health status, particularly in capybaras.

2022 ◽  
Vol 12 (2) ◽  
pp. 335-345
Xiaoyan Zhang ◽  
Wei Zhu ◽  
Junjie Lu

MicroRNAs (miRNAs/miRs) have been identified to serve a key role in the development of tumors. However, the role of miR-133b in colorectal cancer (CRC) remains largely unclear. This study will investigate the role and mechanism of miR-133b in CRC. Reverse transcription-quantitative polymerase chain reaction analysis was performed to detect the level of miR-133b in CRC cell lines. Bioinformatics software TargetScan predicted the potential target genes of miR-133b, and a dual luciferase reporter assay was used to confirm this. To investigate the role of miR-133b in CRC cells, miR-133b was upregulated or downregulated in CRC cell lines (SW620 and HT-29) by transfecting with a miR-133b mimic or inhibitor, respectively. Subsequently, cell viability was analyzed using MTT assay, whereas cell apoptosis and the cell cycle distribution were analyzed by flow cytometry. In addition, the associated protein levels were detected using western blot analysis. The results demonstrated that miR-133b was significantly downregulated in CRC cell lines when compared with the normal colonic epithelial NCM-460 cell line. Human antigen R (HuR; also termed ELAVL1) was demonstrated to be a direct target of miR-133b and was negatively regulated by miR-133b. HuR was also notably upregulated in the CRC cell lines when compared with the normal control. Transfection of SW620 and HT-29 cells with the miR-133b mimic significantly inhibited cell viability, and induced cell apoptosis and G1 phase arrest, while upregulation of HuR demonstrated the opposite effects. Furthermore, the present data demonstrated that the miR-133b mimic significantly enhanced the protein levels of p21 and p27, and downregulated cyclin D1 and cyclin A levels in SW620 and HT-29 cells; the opposite effects were observed following treatment with the miR-133b inhibitor. In conclusion, the data indicate that miR-133b suppressed CRC cell growth by targeting HuR.

Metals ◽  
2022 ◽  
Vol 12 (1) ◽  
pp. 148
Abdulaziz A. Al-Khedhairy ◽  
Rizwan Wahab

Cancer is a cataclysmic disease that affects not only the target organ, but also the whole body. Metal-based nanoparticles (NPs) have recently emerged as a better option for the treatment of this deadly disease. Accordingly, the present work describes a means to control the growth of cancer cells by using colloidal silver nanoparticles (AgNPs) processed via homemade solutions and the characterization of these materials. The AgNPs may become an instantaneous solution for the treatment of these deadly diseases and to minimize or remove these problems. The AgNPs exhibit excellent control of the growth rate of human liver (HepG2) and breast (MCF-7) cancer cells, even at a very low concentrations. The cytotoxic effects of AgNPs on HepG2 and MCF-7 cancer cells were dose dependent (2–200 μg/mL), as evaluated using MTT and NRU assays. The production of reactive oxygen species (ROS) was increased by 136% and 142% in HepG2 and MCF-7 cells treated with AgNPs, respectively. The quantitative polymerase chain reaction (qPCR) data for both cell types (HepG2 and MCF-7) after exposure to AgNPs showed up- and downregulation of the expression of apoptotic (p53, Bax, caspase-3) and anti-apoptotic (BCl2) genes; moreover, their roles were described. This work shows that NPs were successfully prepared and controlled the growth of both types of cancer cells.

Nutrients ◽  
2022 ◽  
Vol 14 (2) ◽  
pp. 353
Lucia Alonso-Pedrero ◽  
Carolina Donat-Vargas ◽  
Maira Bes-Rastrollo ◽  
Ana Ojeda-Rodríguez ◽  
Guillermo Zalba ◽  

Exposure to persistent organic pollutants (POPs) may influence telomere length (TL), which is considered as a marker of biological age associated with the risk of chronic disease. We hypothesized that dietary exposure to polychlorinated biphenyls (PCBs) and dioxins could affect TL. Our aim was to evaluate the association of dietary exposure to PCBs and dioxins with TL. In this cross-sectional study of 886 subjects older than 55 y (mean age: 67.7; standard deviation (SD): 6.1; 27% women) from the “Seguimiento Universidad de Navarra” (SUN) project. TL was determined by real-time quantitative polymerase chain reaction and dietary PCBs and dioxins exposure was collected using a validated 136-item Food Frequency Questionnaire. Multivariable linear regression models were used to control for potential confounding factors. Shorter TL was associated with dietary total PCBs (SD of T/S ratio/(ng/day) = −0.30 × 10−7; 95% CI, −0.55 × 10−7 to −0.06 × 10−7), dioxin-like PCBs (DL-PCBs) (SD of T/S ratio/(pg WHO TEQ (Toxic Equivalents)/day) = −6.17 × 10−7; 95% CI, −11.30 × 10−7 to −1.03 × 10−7), and total TEQ exposure (SD of T/S ratio/(pg WHO TEQ/day) = −5.02 × 10−7; 95% CI, −9.44 × 10−7 to −0.61 × 10−7), but not with dioxins (SD of T/S ratio/(pg WHO TEQ/day) = −13.90 × 10−7; 95% CI, −37.70 × 10−7 to 9.79 × 10−7). In this sample of middle-aged and older Spanish adults, dietary exposure to total PCBs and DL-PCBs alone and together with dioxins was associated with shorter TL. Further longitudinal studies, preferably with POPs measured in biological samples, are needed to confirm this finding.

PLoS ONE ◽  
2022 ◽  
Vol 17 (1) ◽  
pp. e0261853
Margaret G. Mills ◽  
Emily Bruce ◽  
Meei-Li Huang ◽  
Jessica W. Crothers ◽  
Ollivier Hyrien ◽  

Reverse transcription–quantitative polymerase chain reaction (RT-qPCR) is used worldwide to test and trace the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). “Extraction-less” or “direct” real time–reverse transcription polymerase chain reaction (RT-PCR) is a transparent and accessible qualitative method for SARS-CoV-2 detection from nasopharyngeal or oral pharyngeal samples with the potential to generate actionable data more quickly, at a lower cost, and with fewer experimental resources than full RT-qPCR. This study engaged 10 global testing sites, including laboratories currently experiencing testing limitations due to reagent or equipment shortages, in an international interlaboratory ring trial. Participating laboratories were provided a common protocol, common reagents, aliquots of identical pooled clinical samples, and purified nucleic acids and used their existing in-house equipment. We observed 100% concordance across laboratories in the correct identification of all positive and negative samples, with highly similar cycle threshold values. The test also performed well when applied to locally collected patient nasopharyngeal samples, provided the viral transport media did not contain charcoal or guanidine, both of which appeared to potently inhibit the RT-PCR reaction. Our results suggest that direct RT-PCR assay methods can be clearly translated across sites utilizing readily available equipment and expertise and are thus a feasible option for more efficient COVID-19 coronavirus disease testing as demanded by the continuing pandemic.

Jing Du ◽  
Benjamin R. Thomson ◽  
Tuncer Onay ◽  
Susan E. Quaggin

Background: Schlemm’s canal (SC) is a large vessel residing in the iridocorneal angle and is required to regulate aqueous humor outflow. Normal SC structure and function is indispensable for maintaining normal intraocular pressure, and elevated intraocular pressure is a risk factor for development of glaucoma. Recent reports have identified a key role of the angiopoietin-Tie2 pathway for SC development and function; however, the role of the orphan receptor Tie1 has not been clarified. Methods: We used Tie1 knock out mice to study the function of Tie1 in SC development and function. Real-time quantitative polymerase chain reaction and Western blot analyses were used to verify Tie1 deletion. High-resolution microscopy of mouse SC whole mount and cross sections were used to study SC morphology. Measurement of intraocular pressure in live mice was used to study the impact of Tie1 on SC function. Results: Tie1 is highly expressed in both human and mouse SC. Tie1 knock out mice display hypomorphic SC and elevated intraocular pressure as a result of attenuated SC development. Conclusions: Tie1 is indispensable for SC development and function, supporting it as a novel target for future SC-targeted glaucoma therapies and a candidate gene for glaucoma in humans.

2022 ◽  
Vol 12 ◽  
Ningning Fu ◽  
Jiaxing Li ◽  
Ming Wang ◽  
Lili Ren ◽  
Shixiang Zong ◽  

A strict relationship exists between the Sirex noctilio and the Amylostereum areolatum, which is carried and spread by its partner. The growth and development of this symbiotic fungus is key to complete the life history of the Sirex woodwasp. Real-time quantitative polymerase chain reaction (RT-qPCR) is used to measure gene expression in samples of A. areolatum at different growth stages and explore the key genes and pathways involved in the growth and development of this symbiotic fungus. To obtain accurate RT-qPCR data, target genes need to be normalized by reference genes that are stably expressed under specific experimental conditions. In our study, the stability of 10 candidate reference genes in symbiotic fungal samples at different growth and development stages was evaluated using geNorm, NormFinder, BestKeeper, delta Ct methods, and RefFinder. Meanwhile, laccase1 was used to validate the stability of the selected reference gene. Under the experimental conditions of this study, p450, CYP, and γ-TUB were identified as suitable reference genes. This work is the first to systematically evaluate the reference genes for RT-qPCR results normalization during the growth of this symbiotic fungus, which lays a foundation for further gene expression experiments and understanding the symbiotic relationship and mechanism between S. noctilio and A. areolatum.

Foods ◽  
2022 ◽  
Vol 11 (2) ◽  
pp. 194
Xin Fan ◽  
Huimin Guo ◽  
Cong Teng ◽  
Biao Zhang ◽  
Christophe Blecker ◽  

Quinoa peptides are the bioactive components obtained from quinoa protein digestion, which have been proved to possess various biological activities. However, there are few studies on the anticancer activity of quinoa peptides, and the mechanism has not been clarified. In this study, the novel quinoa peptides were obtained from quinoa protein hydrolysate and identified by liquid chromatography–tandem mass spectrometry (LC–MS/MS). The anticancer activity of these peptides was predicted by PeptideRanker and evaluated using an antiproliferative assay in colon cancer Caco-2 cells. Combined with the result of histone deacetylase 1 (HDAC1) inhibitory activity assay, the highly anticancer activity peptides FHPFPR, NWFPLPR, and HYNPYFPG were screened and further investigated. Molecular docking was used to analyze the binding site between peptides and HDAC1, and results showed that three peptides were bound in the active pocket of HDAC1. Moreover, real-time quantitative polymerase chain reaction (RT-qPCR), and Western blot showed that the expression of HDAC1, NFκB, IL-6, IL-8, Bcl-2 was significantly decreased, whereas caspase3 expression showed a remarkable evaluation. In conclusion, quinoa peptides may have the potential to protect against cancer development by inhibiting HDAC1 activity and regulating the expression of the cancer-related genes, which indicates that these peptides could be explored as functional foods to alleviate colon cancer.

Plant Disease ◽  
2022 ◽  
Madalyn Shires ◽  
Alice Wright ◽  
Scott Harper

Little cherry virus-2 (LChV-2) is a viral pathogen that is reaching epidemic levels in Washington state. This virus is insect-vectored and has significant impacts on sweet cherry production. To aid growers in making informed management decisions we sought to develop a diagnostic assay to better detect isolates of LChV-2 currently found in Washington, allowing for more accurate estimations of disease occurrence. This study showed that there were two distinct genotypes of LChV-2 present in Washington state. This information was used to develop an up-to-date reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR) assay, which was then optimized, validated, and compared to four previously published assays using a panel of field samples. This comparison demonstrated that the newly developed assay provided greater sensitivity, accurately detecting less than 10 copies per reaction and could detect both LChV-2 genotypes. Finally, we examined the effect of potential inhibitors in various tissue types from cherry, finding that young leaf tissue affected sensitivity of detection less than root tissues.

2022 ◽  
Ruyi Zhang ◽  
Jiangbo Du ◽  
Zhendong Xiao ◽  
Yuan Jiang ◽  
Qiao Weng ◽  

Abstract Purpose To explore changes of Telomere length (TL) and mitochondrial copy number (mtDNA-CN) in preeclampsia (PE) and to evaluatethe combined effect of maternal TL and mtDNA-CN on PE risk.Methods A case-control study of 471 subjects (130 PE cases and 341 age frequency matched controls) was conducted in Nanjing Drum Tower Hospital, Jiangsu Province of China. Relative telomere length (RTL) and mtDNA-CN were measured using quantitative polymerase chain reaction (qPCR) and PE risk was calculated between groups by logistic regression analyses.Results PE patients displayed longer RTL (0.48 versus 0.30) and higher mtDNA-CN (3.02 versus 2.00) in maternal bloodas well as longer cord blood RTL(0.61 versus 0.35) but lower mtDNA-CN (1.69 versus 5.49) in cord blood (all p<0.001). Exercise during pregnancy exerted an obvious effect of prolonging maternal telomere length. Multiparous, women with folic acid intake during early pregnancy and those delivered vaginally showed longer telomere length while those factors imposed no or opposite effect on RTL in PE cases. Furthermore, RTL and mtDNA-CN were positively correlated in controls (in maternal blood r=0.18, p<0.01; in cord blood r=0.19, p<0.001), but this correlation was disrupted in PE cases, no matter in maternal blood or in cord blood. Longer maternal RTL and higher mtDNA-CN were associated with higher risk of PE, and the ROC curve of RTL and mtDNA-CN in predicting PE risk presented an AUC of 0.755(95%CI: 0.698-0.812).Conclusions Interaction of TL and mtDNA-CN may play an important role in pathogenesis of PE and it could be a potential biomarker indicating PE risk.

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