Chromatin Immunoprecipitation and Quantitative Real-Time PCR to Assess Binding of a Protein of Interest to Identified Predicted Binding Sites Within a Promoter

2017 ◽  
pp. 23-32
Author(s):  
Jordan E. Read
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Chromatin Immunoprecipitation ◽  
Binding Sites ◽  
Quantitative Real Time Pcr

scholarly journals KLK11 promotes the activation of mTOR and protein synthesis to facilitate cardiac hypertrophy

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Yi Wang ◽  
Hongjuan Liao ◽  
Yueheng Wang ◽  
Jinlin Zhou ◽  
Feng Wang ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Angiotensin Ii ◽  
Cardiac Hypertrophy ◽  
Western Blot ◽  
Real Time ◽  
Real Time Pcr ◽  
Mtor Signaling ◽  
Heart Weight ◽  
Cardiomyocyte Hypertrophy ◽  
Quantitative Real Time Pcr

Abstract Background Cardiovascular diseases have become the leading cause of death worldwide, and cardiac hypertrophy is the core mechanism underlying cardiac defect and heart failure. However, the underlying mechanisms of cardiac hypertrophy are not fully understood. Here we investigated the roles of Kallikrein 11 (KLK11) in cardiac hypertrophy. Methods Human and mouse hypertrophic heart tissues were used to determine the expression of KLK11 with quantitative real-time PCR and western blot. Mouse cardiac hypertrophy was induced by transverse aortic constriction (TAC), and cardiomyocyte hypertrophy was induced by angiotensin II. Cardiac function was analyzed by echocardiography. The signaling pathway was analyzed by western blot. Protein synthesis was monitored by the incorporation of [3H]-leucine. Gene expression was analyzed by quantitative real-time PCR. Results The mRNA and protein levels of KLK11 were upregulated in human hypertrophic hearts. We also induced cardiac hypertrophy in mice and observed the upregulation of KLK11 in hypertrophic hearts. Our in vitro experiments demonstrated that KLK11 overexpression promoted whereas KLK11 knockdown repressed cardiomyocytes hypertrophy induced by angiotensin II, as evidenced by cardiomyocyte size and the expression of hypertrophy-related fetal genes. Besides, we knocked down KLK11 expression in mouse hearts with adeno-associated virus 9. Knockdown of KLK11 in mouse hearts inhibited TAC-induced decline in fraction shortening and ejection fraction, reduced the increase in heart weight, cardiomyocyte size, and expression of hypertrophic fetal genes. We also observed that KLK11 promoted protein synthesis, the key feature of cardiomyocyte hypertrophy, by regulating the pivotal machines S6K1 and 4EBP1. Mechanism study demonstrated that KLK11 promoted the activation of AKT-mTOR signaling to promote S6K1 and 4EBP1 pathway and protein synthesis. Repression of mTOR with rapamycin blocked the effects of KLK11 on S6K1 and 4EBP1 as well as protein synthesis. Besides, rapamycin treatment blocked the roles of KLK11 in the regulation of cardiomyocyte hypertrophy. Conclusions Our findings demonstrated that KLK11 promoted cardiomyocyte hypertrophy by activating AKT-mTOR signaling to promote protein synthesis.

Quantitative real-time PCR assay to detect transgene copy number in cotton (Gossypium hirsutum)

2008 ◽  
Vol 375 (1) ◽  
pp. 150-152 ◽  
Author(s):  
Cheng Xin Yi ◽  
Jun Zhang ◽  
Ka Man Chan ◽  
Xiao Kun Liu ◽  
Yan Hong
Keyword(s):  
Gossypium Hirsutum ◽  
Real Time ◽  
Real Time Pcr ◽  
Copy Number ◽  
Quantitative Real Time Pcr ◽  
Pcr Assay ◽  
Transgene Copy Number

scholarly journals rpoB and efp are stable candidate reference genes for quantitative real-time PCR analysis in Saccharopolyspora spinosa

2021 ◽  
Vol 35 (1) ◽  
pp. 619-632
Author(s):  
Xiaomeng Liu ◽  
Yunpeng Zhang ◽  
Kexue Huang ◽  
Tie Yin ◽  
Qi Li ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Reference Genes ◽  
Pcr Analysis ◽  
Quantitative Real Time Pcr ◽  
Saccharopolyspora Spinosa

scholarly journals Virological Diagnosis of Herpes Simplex Virus 1 Esophagitis by Quantitative Real-Time PCR Assay

2011 ◽  
Vol 50 (3) ◽  
pp. 948-952 ◽  
Author(s):  
J.-F. Jazeron ◽  
C. Barbe ◽  
E. Frobert ◽  
F. Renois ◽  
D. Talmud ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Real Time ◽  
Real Time Pcr ◽  
Quantitative Real Time Pcr ◽  
Pcr Assay ◽  
Herpes Simplex Virus 1 ◽  
Virological Diagnosis ◽  
Simplex Virus

scholarly journals Regulation of virulence and β-lactamase gene expression in Staphylococcus aureus isolates: cooperation of two-component systems in bloodstream superbugs

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Sanaz Dehbashi ◽  
Hamed Tahmasebi ◽  
Behrouz Zeyni ◽  
Mohammad Reza Arabestani
Keyword(s):  
Staphylococcus Aureus ◽  
Real Time ◽  
Real Time Pcr ◽  
Quantitative Real Time Pcr ◽  
Gene Expressions ◽  
Resistance Development ◽  
Component Systems ◽  
Two Component ◽  
Two Component Systems ◽  
Lactamase Gene

Abstract Background Methicillin-resistant Staphylococcus aureus (MRSA)-bloodstream infections (BSI) are predominantly seen in the hospital or healthcare-associated host. Nevertheless, the interactions of virulence factor (VFs) regulators and β-lactam resistance in MRSA-BSI are unclear. This study aims to characterize the molecular relationship of two-component systems of VFs and the expression of the β-lactamase gene in MRSA-BSI isolates. In this study, 639 samples were collected from BSI and identified by phenotypic methods. We performed extensive molecular characterization, including SCCmec type, agr type, VFs gene profiles determinations, and MLST on isolates. Also, a quantitative real-time PCR (q-RT PCR) assay was developed for identifying the gene expressions. Results Ninety-one (91) S. aureus and 61 MRSA (67.0%) strains were detected in BSI samples. The presence of VFs and SCCmec genes in MRSA isolates were as follows: tst (31.4%), etA (18.0%), etB (8.19%), lukS-PVL (31.4%), lukF-PV (18.0%), lukE-lukD (16.3%), edin (3.2%), hla (16.3%), hlb (18.0%), hld (14.7%), hlg (22.9%), SCCmecI (16.3%), SCCmecII (22.9%), SCCmecIII (36.0%), SCCmecIV (21.3%), and SCCmecV (16.3%). Quantitative real-time PCR showed overexpression of mecRI and mecI in the toxigenic isolates. Moreover, RNAIII and sarA genes were the highest expressions of MRSA strains. The multi-locus sequence typing data confirmed a high prevalence of CC5, CC8, and CC30. However, ST30, ST22, and ST5 were the most prevalent in the resistant and toxigenic strains. Conclusion We demonstrated that although regulation of β-lactamase gene expressions is a significant contributor to resistance development, two-component systems also influence antibiotic resistance development in MRSA-BSI isolates. This indicates that resistant strains might have pathogenic potential. We also confirmed that some MLST types are more successful colonizers with a potential for MRSA-BSI.

Diagnosis of neuroschistosomiasis by antibody specificity index and semi-quantitative real-time PCR from cerebrospinal fluid and serum

2014 ◽  
Vol 63 (2) ◽  
pp. 309-312 ◽  
Author(s):  
Georg Härter ◽  
Hagen Frickmann ◽  
Sebastian Zenk ◽  
Dominic Wichmann ◽  
Bettina Ammann ◽  
...  
Keyword(s):  
Cerebrospinal Fluid ◽  
Real Time ◽  
Real Time Pcr ◽  
Clinical Symptoms ◽  
Diagnostic Procedures ◽  
Lower Limbs ◽  
Antibody Specificity ◽  
Quantitative Real Time Pcr ◽  
Specific Antibodies ◽  
Routine Diagnosis

We describe the case of a 16-year-old German male expatriate from Ghana who presented with obstipation, dysuria, dysaesthesia of the gluteal region and the lower limbs, bilateral plantar hypaesthesia and paraesthesia without pareses. A serum–cerebrospinal fluid (CSF) Schistosoma spp. specific antibody specificity index of 3.1 was considered highly suggestive of intrathecal synthesis of anti-Schistosoma spp. specific antibodies, although standardization of this procedure has not previously been described. Diagnosis was confirmed by detection of Schistosoma DNA in CSF by semi-quantitative real-time PCR at 100-fold concentration compared with serum. Accordingly the two diagnostic procedures, which have not previously been applied for routine diagnosis, appear to be useful for the diagnosis of neuroschistosomiasis. Clinical symptoms resolved following anthelmintic and anti-inflammatory therapy.

Sign in / Sign up

Export Citation Format

Share Document