scholarly journals Quantitative analysis method of mRNA expression using dual RNA in situ hybridization by comparison with housekeeping genes v1 (protocols.io.badcia2w)

protocols.io ◽  
2019 ◽  
Author(s):  
Byung Joon
Keyword(s):  
In Situ Hybridization ◽  
Quantitative Analysis ◽  
Mrna Expression ◽  
Housekeeping Genes ◽  
Analysis Method ◽  
Quantitative Analysis Method ◽  
Rna In Situ Hybridization

A Direct Quantitative Raman Method for the Measurement of Dissolved Bisulfate in Acid-Sulfate Fluids

2018 ◽  
Vol 72 (8) ◽  
pp. 1234-1243 ◽  
Author(s):  
Shichuan Xi ◽  
Xin Zhang ◽  
Zhendong Luan ◽  
Zengfeng Du ◽  
Lianfu Li ◽  
...  
Keyword(s):  
Quantitative Analysis ◽  
Physicochemical Parameters ◽  
Water Molecules ◽  
Analysis Method ◽  
Hydrogen Phosphate ◽  
Acid Sulfate ◽  
Different Temperatures ◽  
Quantitative Analysis Method ◽  
In Situ Detection

Raman spectroscopy has been applied to the quantitative analysis of the concentration of bisulfate in acid-sulfate fluids at different temperatures. The quantitative analysis method is based on the peak area ratios of [Formula: see text](ν1) and H2O (ν2), where PA([Formula: see text]/H2O) = [[Formula: see text]] × (0.0066 × T + 1.3070) at a temperature range of 0–100 ℃. We found that the molal scattering coefficient of bisulfate increases slightly at the elevated temperature may be due to the changes of fraction of water molecules that are hydrogen-bonded. The method can also be applied to analyze physicochemical parameters of other acid fluids, such as hydrogen phosphate, bicarbonate, etc., and especially to the in situ detection of deep sea acid-sulfate hydrothermal fluids in the future.

scholarly journals Association of Matrix Metalloproteinase-2 mRNA Expression with Subtypes of Pediatric Cholesteatoma

2021 ◽  
Vol 2021 ◽  
pp. 1-8
Author(s):  
Taichi Kan ◽  
Hiromi Ueda ◽  
Taishi Takahara ◽  
Yoshimasa Tsuchiya ◽  
Mayuko Kishimoto ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Matrix Metalloproteinase ◽  
Mrna Expression ◽  
Bone Destruction ◽  
Expression Level ◽  
Matrix Metalloproteinase 2 ◽  
Mrna Expression Level ◽  
Congenital Cholesteatoma ◽  
Rna In Situ Hybridization

Objective. Cholesteatoma is a clinically heterogeneous disease, with some patients showing spontaneous regression, while others experiencing an aggressive, lethal disease. Cholesteatoma in children can be divided into two types: congenital and acquired. Identifying good prognostic markers is needed to help select patients who will require immediate surgical intervention. Matrix metalloproteinase-2 (MMP2) was previously reported to play an important role in cholesteatoma progression, by promoting bone destruction and keratinocyte infiltration. Herein, we analyzed MMP2 mRNA expression level in cholesteatoma using RNA-in situ hybridization in formalin-fixed, paraffin-embedded (FFPE) tissue samples. Methods. Sixty patients with cholesteatoma under 15 years old, who underwent their primary surgery at Aichi Medical University’s Otolaryngology Department, were analyzed for MMP2 expression level, using RNA-in situ hybridization. Results. There were no significant differences in MMP2 mRNA expression level between congenital cholesteatoma and acquired cholesteatomas. In congenital cholesteatoma, higher MMP2 signals were observed in the open type than in the closed type ( p < 0.001 ). In acquired cholesteatoma, higher MMP2 signals were observed in the pars tensa than in the pars flaccida ( p < 0.001 ). MMP2 mRNA expression level was almost exclusively found in the fibroblasts or in the inflammatory cells in the stroma, but not in the epithelium. Conclusion. Our study reveals that MMP2 mRNA expression level is strongly associated with the subtypes of cholesteatoma. The findings suggest that the level of expression of MMP2 mRNA may be related to the pathogenesis and aggressive features of cholesteatoma.

scholarly journals Development of Antibodies against HPV-6 and HPV-11 for the Study of Laryngeal Papilloma

Viruses ◽  
2021 ◽  
Vol 13 (10) ◽  
pp. 2024
Author(s):  
Taro Ikegami ◽  
Norimoto Kise ◽  
Hidetoshi Kinjyo ◽  
Shunsuke Kondo ◽  
Mikio Suzuki ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Mrna Expression ◽  
Positive Reaction ◽  
Viral Gene Expression ◽  
Viral Gene ◽  
Laryngeal Papilloma ◽  
Hpv Dna ◽  
Functional Studies ◽  
Rna In Situ Hybridization

Laryngeal papilloma (LP), which is associated with infection by human papillomavirus (HPV)-6 or -11, displays aggressive growth. The precise molecular mechanism underlying the tumorigenesis of LP has yet to be uncovered. Building on our earlier research into HPV-6, in this study, the viral gene expression of HPV-11 was investigated by quantitative PCR and DNA/RNA in situ hybridization. Additionally, newly developed antibodies against the E4 protein of HPV-6 and HPV-11 were evaluated by immunohistochemistry. The average viral load of HPV-11 in LP was 1.95 ± 0.66 × 105 copies/ng DNA, and 88% of HPV mRNA expression was found to be E4, E5a, and E5b mRNAs. According to RNA in situ hybridization, E4 and E5b mRNAs were expressed from the middle to upper part of the epithelium. E4 immunohistochemistry revealed a wide positive reaction in the upper cell layer in line with E4 mRNA expression. Other head and neck lesions with HPV-11 infection also showed a positive reaction in E4 immunohistochemistry. The distribution pattern of HPV DNA, viral mRNA, and E4 protein in LP with HPV-11 infection was quite similar to that of HPV-6. Therefore, it might be possible to apply these E4-specific antibodies in other functional studies as well as clinical applications, including targeted molecular therapies in patients with HPV-6 and HPV-11 infection.

scholarly journals Higher Tissue Factor (TF) Expression in the Lungs of COVID-19 Pneumonia Patients Than Patients with Acute Respiratory Distress Syndrome: Association with Thrombi Formation

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 4-4
Author(s):  
Sandeep Subrahmanian ◽  
Alain Borczuk ◽  
Steven P. Salvatore ◽  
Jeffrey Laurence ◽  
Jasimuddin Ahamed
Keyword(s):  
Mechanical Ventilation ◽  
In Situ Hybridization ◽  
Mrna Expression ◽  
Tissue Factor ◽  
Fluorescence Probes ◽  
Coagulation Cascade ◽  
Extrinsic Factors ◽  
Platelet Factor ◽  
Rna In Situ Hybridization

A substantial proportion of patients with severe COVID-19 pneumonia develop thrombosis (both venous and arterial) via an undefined mechanism. Systemic elevation of high levels of D-dimer, a marker of coagulation activation and thrombolysis, is evident in almost all advanced COVID-19 patients and is associated with disease severity and mortality. However, the extrinsic factors that initiate blood coagulation in COVID-19 is not clear. Because tissue factor (TF) is the prime initiator of the extrinsic coagulation cascade, and because it is expressed and exposed under inflammatory conditions leading to vascular damage, we tested the hypothesis that higher TF expression is responsible for thrombi formation in the lungs of patients with severe COVID-19. To this end, we examined autopsy lung tissues from five COVID-19 pneumonia patients with acute respiratory syndrome (ARDS) who required ICU hospitalization, with 4 of the 5 patients requiring mechanical ventilation before they died, and five controls with acute ARDS caused by bacterial pneumonia, one with a prior influenza infection, and all on mechanical ventilation. Histological findings revealed all the COVID-19 lungs had characteristics of ARDS, including DAD with hyaline deposition and inflammatory cell invasion. Immunofluorescence staining showed TF expression throughout all lung tissues and in many blood vessels that were filled with thrombi with either fibrin, activated platelets, or both. TF expression was significantly higher in COVID-19 than control ARDS lung tissues (1.2 ± 0.3% in COVID-19 vs. 0.52 ± 0.2% in ARDS controls (p=0.004). Fibrin-enriched thrombi areas were higher in COVID-19 cases than in controls (1.6 ± 0.36% vs. 0.94 ± 0.4%; p=0.008) and correlated with TF expression (R2=0.4, p=0.02). Platelet factor 4 (PF4)-enriched thrombi areas were also higher in COVID-19 lung, but this trend was not statistically significant (0.94 ± 0.4% in COVID-19 and 0.54 ± 0.3% in controls; p=0.09), although it did, however, correlate with TF expression (R2=0.4, p=0.02). Many thrombi were in close proximity to TF-expressing areas in both COVID-19 and ARDS pneumonia controls. Dual RNA in situ hybridization with SARS-CoV-2 and TF fluorescence probes showed variable viral and TF mRNA expression. Increased TF mRNA expression was seen in COVID-19 vs. control lung (0.77 ± 0.4 % vs. 0.31 ± 0.15 %, p=0.05). TF mRNA expression correlated with viral mRNA in COVID-19 patients (R2=0.78, p=0.01). High-resolution images identified both sporadic and clustered SARS-CoV-2, and some areas co-localized with TF mRNA expression. We conclude that higher TF expression might be responsible for fibrin formation and platelet activation in the lungs of both COVID-19 and ARDS controls. Our observation of higher TF expression in COVID-19 patients was documented by two very sensitive methods, RNA in situ hybridization and immunostaining with very specific antibodies against TF and mRNA probes. Its correlation with SARS-CoV-2 mRNA suggests that SARS-CoV-2 infection induces both de novo gene transcription and protein synthesis in the lungs of COVID-19 patients. Thus, TF-initiated extrinsic coagulation might be responsible for the critical thrombi formation observed in many COVID-19 cases, rendering TF a potential therapeutic target. Disclosures Laurence: Alexion Pharmaceuticals: Honoraria, Research Funding.

scholarly journals RNA In Situ Hybridization for Detecting Gene Expression Patterns in the Abdomens and Wings of Drosophila Species

2021 ◽  
Vol 4 (1) ◽  
pp. 20
Author(s):  
Mujeeb Shittu ◽  
Tessa Steenwinkel ◽  
William Dion ◽  
Nathan Ostlund ◽  
Komal Raja ◽  
...  
Keyword(s):  
Gene Expression ◽  
In Situ Hybridization ◽  
Expression Patterns ◽  
Drosophila Species ◽  
Gene Expression Patterns ◽  
Probe Design ◽  
Tissue Preparation ◽  
Design And Synthesis ◽  
Rna In Situ Hybridization

RNA in situ hybridization (ISH) is used to visualize spatio-temporal gene expression patterns with broad applications in biology and biomedicine. Here we provide a protocol for mRNA ISH in developing pupal wings and abdomens for model and non-model Drosophila species. We describe best practices in pupal staging, tissue preparation, probe design and synthesis, imaging of gene expression patterns, and image-editing techniques. This protocol has been successfully used to investigate the roles of genes underlying the evolution of novel color patterns in non-model Drosophila species.

Sign in / Sign up

Export Citation Format

Share Document