scholarly journals Extraction and reverse transcription of total rna from mouse brain-derived endothelial cells.3 infected by streptococcus Suis 2

2021 ◽  
pp. 39-41
Author(s):  
Mingcheng Liu ◽  
Oksana Kasianenko
Keyword(s):  
Gene Expression ◽  
Endothelial Cells ◽  
Protein Expression ◽  
Mouse Brain ◽  
Reverse Transcription ◽  
Rna Extraction ◽  
Streptococcus Suis ◽  
High Quality ◽  
Total Rna ◽  
Polymerase Chain

Streptococcus suis 2 is an important emerging zoonotic pathogen. It mainly causes meningitis in pigs. We use SS2 to infect bEnd.3 to get stable cDNA for next research on differences in gene expression and protein expression of cytokines. The paper presents an SS2 study for bEnd.3 infection to obtain stable cDNA for subsequent study of differences in gene expression and cytokine protein expression. Objective: The aim of this study was to extract the total RNA from mouse brain-derived Endothelial cells (bEnd.3) infected by Streptococcus suis serotype 2 (SS2) and transcript to complementary DNA (cDNA). Materials and methods: SS2 strain were obtained from Jilin University, China. BEnd.3 was from Henan institute of Science of Technology, China. Reverse transcription kit was from Takara company, Japan. Trizol was from Bioteke company,China. Nanodrop instrument was from Thermo company, USA. Polymerase chain reaction (PCR) instrument was from Biometra company, Germany. We used SS2 to infect bEnd.3 at a multiplicity of infection (MOI) of 100 for 12h. Cells were harvested and Trizol method was chose to extract the total RNA of bEnd.3 infected by SS2. Nanodrop instrument was used to measure the concentration of RNA and the values of OD260/280 and OD260/230. RNA were transcripted to cDNA with reverse transcription kit by PCR instrument. Results: trizol method used in this study was reliable and high-quality RNA were obtained. Stable cDNA were obtained by reverse transcription kit. Conclusion: in this experiment high-quality RNA was obtained and reverse transcribed to stable cDNA for subsequent detection of related cytokines. This study provides an approximate RNA extraction method and good experimental foundation for downstream research.

Isolation of high-quality total RNA from rumen anaerobic bacteria and fungi, and subsequent detection of glycoside hydrolases

2011 ◽  
Vol 57 (7) ◽  
pp. 590-598 ◽  
Author(s):  
Pan Wang ◽  
Meng Qi ◽  
Perry Barboza ◽  
Mary Beth Leigh ◽  
Emilio Ungerfeld ◽  
...  
Keyword(s):  
Gene Expression ◽  
Solid Phase ◽  
Large Subunit ◽  
Rna Extraction ◽  
Small Subunit ◽  
Glycoside Hydrolases ◽  
Small Subunit Rrna ◽  
High Quality ◽  
Major Barrier ◽  
Total Rna

The rumen is one of the most powerful fibrolytic fermentation systems known. Gene expression analyses, such as reverse transcription PCR (RT-PCR), microarrays, and metatranscriptomics, are techniques that could significantly expand our understanding of this ecosystem. The ability to isolate and stabilize representative RNA samples is critical to obtaining reliable results with these procedures. In this study, we successfully isolated high-quality total RNA from the solid phase of ruminal contents by using an improved RNA extraction method. This method is based on liquid nitrogen grinding of whole ruminal solids without microbial detachment and acid guanidinium – phenol – chloroform extraction combined with column purification. Yields of total RNA were as high as 150 µg per g of fresh ruminal content. The typical large subunit/small subunit rRNA ratio ranged from 1.8 to 2.0 with an RNA integrity number (Agilent Technologies) greater than 8.5. By eliminating the detachment step, the resulting RNA was more representative of the complete ecosystem. Our improved method removed a major barrier limiting analysis of rumen microbial function from a gene expression perspective. The polyA-tailed eukaryotic mRNAs obtained have successfully been applied to next-generation sequencing, and metatranscriptomic analysis of the solid fraction of rumen contents revealed abundant sequences related to rumen fungi.

scholarly journals High-quality total RNA isolation from melon (Cucumis melo L.) fruits rich in polysaccharides

2017 ◽  
Vol 38 (4) ◽  
pp. 2201 ◽  
Author(s):  
Gabrielle Silveira de Campos ◽  
Ricardo Antônio Ayub ◽  
Rafael Mazer Etto ◽  
Carolina Weigert Galvão ◽  
Marília Aparecida Stroka ◽  
...  
Keyword(s):  
Rna Extraction ◽  
Sodium Perchlorate ◽  
Rna Isolation ◽  
World Market ◽  
Molecular Techniques ◽  
Guanidine Thiocyanate ◽  
High Quality ◽  
Total Rna ◽  
High Concentrations ◽  
Cucumis Melo L

Melon, a member of the family Cucurbitaceae, is the fourth most important fruit in the world market and, on a volume basis, is Brazil’s main fresh fruit export. Many molecular techniques used to understand the maturation of these fruits require high concentrations of highly purified RNA. However, melons are rich in polyphenolic compounds and polysaccharides, which interfere with RNA extraction. This study aimed to determine the most appropriate method for total RNA extraction from melon fruits. Six extraction buffers were tested: T1) guanidine thiocyanate/phenol/chloroform; T2) sodium azide/?-mercaptoethanol; T3) phenol/guanidine thiocyanate; T4) CTAB/PVP/?-mercaptoethanol; T5) SDS/sodium perchlorate/PVP/?-mercaptoethanol, and T6) sarkosyl/PVP/guanidine thiocyanate, using the AxyPrepTM Multisource Total RNA Miniprep Kit. The best method for extracting RNA from both mature and green fruit was based on the SDS/PVP/?-mercaptoethanol buffer, because it rapidly generated a high quality and quantity of material. In general, higher amounts of RNA were obtained from green than mature fruits, probably due to the lower concentration of polysaccharides and water. The purified material can be used as a template in molecular techniques, such as microarrays, RT-PCR, and in the construction of cDNA and RNA-seq data.

scholarly journals Study of the Alteration of Gene Expression in Adipose Tissue of Diet-Induced Obese Mice by Microarray and Reverse Transcription-Polymerase Chain Reaction Analyses

Endocrinology ◽  
2003 ◽  
Vol 144 (11) ◽  
pp. 4773-4782 ◽  
Author(s):  
R. C. Moraes ◽  
A. Blondet ◽  
K. Birkenkamp-Demtroeder ◽  
J. Tirard ◽  
T. F. Orntoft ◽  
...  
Keyword(s):  
Gene Expression ◽  
Polymerase Chain Reaction ◽  
Adipose Tissue ◽  
Reverse Transcription ◽  
Obese Mice ◽  
Chain Reaction ◽  
Polymerase Chain

G-protein Coupled Estrogen Receptor Expression in Growth Hormone Secreting and Non-Functioning Adenomas

2020 ◽  
Author(s):  
Hande Mefkure Ozkaya ◽  
Muge Sayitoglu ◽  
Nil Comunoglu ◽  
Eda Sun ◽  
Fatma Ela Keskin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Estrogen Receptor ◽  
Protein Expression ◽  
G Protein ◽  
Estrogen Receptor Α ◽  
Receptor Expression ◽  
Positive Correlation ◽  
Polymerase Chain ◽  
G Protein Coupled ◽  
Transforming Gene

Abstract Purpose To evaluate the expression of G-protein coupled estrogen receptor (GPER1), aromatase, estrogen receptor α (ERα), estrogen receptor β (ERβ), pituitary tumor transforming gene (PTTG), and fibroblast growth factor 2 (FGF2) in GH-secreting and non-functioning adenomas (NFA). Methods Thirty patients with acromegaly and 27 patients with NFA were included. Gene expression was determined via quantitative reverse transcription polymerase chain reaction (QRT-PCR). Protein expression was determined via immunohistochemistry. Results There was no difference, in terms of gene expression of aromatase, ERα, PTTG, and FGF2 between the two groups (p>0.05 for all). ERβ gene expression was higher and GPER1 gene expression was lower in GH-secreting adenomas than NFAs (p<0.05 for all). Aromatase and ERβ protein expression was higher in GH-secreting adenomas than NFAs (p=0.01). None of the tumors expressed ERα. GPER1 expression was detected in 62.2% of the GH-secreting adenomas and 45% of NFAs. There was no difference in terms of GPER1, PTTG, FGF2 H scores between the two groups (p>0.05 for all). GPER1 gene expression was positively correlated to ERα, ERβ, PTTG, and FGF2 gene expression (p<0.05 for all). There was a positive correlation between aromatase and GPER1 protein expression (r=0.31; p=0.04). Conclusions GPER1 is expressed at both gene and protein level in a substantial portion of GH-secreting adenomas and NFAs. The finding of a positive correlation between GPER1 and ERα, ERβ, PTTG, and FGF2 gene expression and aromatase and GPER1 protein expression suggests GPER1 along with aromatase and classical ERs might mediate the effects of estrogen through upregulation of PTTG and FGF2.

scholarly journals Protocol adjustment improves the extraction of high-quality total RNA from common bean stems infected by Sclerotinia sclerotiorum

2019 ◽  
Vol 43 ◽  
Author(s):  
Rafael Novais de Miranda ◽  
Caroline Marcela da Silva ◽  
Antonio Carlos da Mota Porto ◽  
Welison Andrade Pereira
Keyword(s):  
Gene Expression ◽  
Common Bean ◽  
Sclerotinia Sclerotiorum ◽  
Rna Extraction ◽  
White Mold ◽  
Basic Requirement ◽  
High Quality ◽  
Commercial Kits ◽  
Made In

ABSTRACT The Straw Test is an assay developed to evaluate the resistance of common bean to white mold, in which the plant stems are inoculated and the symptoms of the disease are monitored. It is plausible to admit that investigating gene expression in pathogen-infected tissues may be strategically interesting. However, obtaining a quality RNA is a basic requirement for this purpose. Therefore, the objective of this study was to evaluate adjustments in protocols of commercial kits in the expectation of improving the quality of RNA obtained from bean stems. For this, plants of two lines were inoculated and the stems pathogen-infected were collected 72 hours after. For RNA extraction, two commercial reagents were used following the manufacturer’s recommendations and then following adaptations in these protocols. In particular, the proposed modifications relate to volumes of supernatant recovered in purification steps, additional step of chloroform purification and extended time for nucleic acids precipitation. The obtained RNA was analyzed by spectrophotometer, electrophoresis and bioanalyzer, then converted into cDNA and subsequently submitted to PCR. From the obtained data, it was observed that the adaptations made in the protocols contributed to better results and that, when the indicative values of RNA quality are guaranteed, the subsequent reactions are more pure, precise and representative.

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