scholarly journals Feasibility of phosphoproteomics on leftover samples after RNA extraction with guanidinium thiocyanate

2021 ◽  
pp. 100078
Author(s):  
Frank Rolfs ◽  
Sander R. Piersma ◽  
Mariana Paes Dias ◽  
Jos Jonkers ◽  
Connie R. Jimenez
Keyword(s):  
Rna Extraction ◽  
Guanidinium Thiocyanate

scholarly journals High Efficiency RNA Extraction From Sperm Cells Using Guanidinium Thiocyanate Supplemented With Tris(2-Carboxyethyl)Phosphine

2021 ◽  
Vol 9 ◽  
Author(s):  
Martin Roszkowski ◽  
Isabelle M. Mansuy
Keyword(s):  
Disulfide Bonds ◽  
High Efficiency ◽  
Rna Extraction ◽  
Limiting Factor ◽  
Sperm Cells ◽  
Biological Assays ◽  
Guanidinium Thiocyanate ◽  
Cell Assays ◽  
Reducing Activity ◽  
Polymerase Chain

The extraction of high-quality ribonucleic acid (RNA) from tissues and cells is a key step in many biological assays. Guanidinium thiocyanate-phenol-chloroform (AGPC) is a widely used and efficient method to obtain pure RNA from most tissues and cells. However, it is not efficient with some cells like sperm cells because they are resistant to chaotropic lysis solutions containing guanidinium thiocyanate such as Buffer RLT+ and Trizol. Here, we show that disulfide bonds are responsible for the chemical resistance of sperm cells to RNA extraction reagents. We show that while β-mercaptoethanol (βME) can increase sperm lysis in Buffer RLT+, it has no effect in Trizol and leaves sperm cells intact. We measured the reduction of disulfide bonds in 2,2′-dithiodipyridine (DTDP) and observed that βME has a pH-dependent activity in chaotropic solutions, suggesting that pH is a limiting factor. We identified tris(2-carboxyethyl)phosphine (TCEP) as an efficient lysis enhancer of AGPC solutions that can retain reducing activity even at acidic pH. Trizol supplemented with TCEP allows the complete and rapid lysis of sperm cells, increasing RNA yield by 100-fold and resulting in RNA with optimal quality for reverse transcription and polymerase chain reaction. Our findings highlight the importance of efficient cell lysis and extraction of various macromolecules for bulk and single-cell assays, and can be applied to other lysis-resistant cells and vesicles, thereby optimizing the amount of required starting material and animals.

scholarly journals MAVRICS: A Robust and Safe Magnetic Nanoparticle based RNA Extraction Method Compatible with Phenol-chloroform Inactivated Infectious Samples v2

2021 ◽  
Author(s):  
Mo Li ◽  
Gerardo Ramos-Mandujano
Keyword(s):  
Extraction Method ◽  
Magnetic Nanoparticle ◽  
Rna Extraction ◽  
Rna Isolation ◽  
Emerging Pathogens ◽  
Nucleic Acid Isolation ◽  
Guanidinium Thiocyanate ◽  
Biology Laboratory ◽  
Alternative Solutions ◽  
Basic Equipment

Diagnosis and surveillance of emerging pathogens such as SARS-CoV-2 depend on nucleic acid isolation from clinical and environmental samples. Under normal circumstances, samples would be processed using commercial proprietary reagents in Biosafety 2 (BSL-2) or higher facilities. A pandemic at the scale of COVID-19 has caused a global shortage of proprietary reagents and BSL-2 laboratories to safely perform testing. Therefore, alternative solutions are urgently needed to address these challenges. We developed an open-source method called Magneticnanoparticle-Aided Viral RNA Isolation of Contagious Samples (MAVRICS) that is built upon reagents that are either readily available or can be synthesized in any molecular biology laboratory with basic equipment. Unlike conventional methods, MAVRICS works directly in samples inactivated in acid guanidinium thiocyanate-phenol-chloroform (e.g., TRIzol), thus allowing infectious samples to be handled safely without biocontainment facilities.

scholarly journals Feasibility of phosphoproteomics on leftover samples after RNA extraction with guanidinium thiocyanate

2020 ◽  
Author(s):  
Frank Rolfs ◽  
Sander R. Piersma ◽  
Mariana Paes Dias ◽  
Jos Jonkers ◽  
Connie R. Jimenez
Keyword(s):  
Large Scale ◽  
Protein Extraction ◽  
Rna Extraction ◽  
Daily Practice ◽  
Extraction Methods ◽  
Small Samples ◽  
Data Types ◽  
Input Material ◽  
Guanidinium Thiocyanate ◽  
Class 1

AbstractIn daily practice, different types of biomolecules are usually extracted for large-scale ‘omics’ analysis with tailored protocols. However, when sample material is limited, an all-in-one strategy is preferable. While lysis of cells and tissues with urea is the accepted standard for phosphoproteomic applications, DNA, RNA and proteins can be simultaneously extracted from small samples using acid guanidinium thiocyanate-phenol-chloroform (AGPC). Use of AGPC for mass spectrometry (MS)-based phosphoproteomics has been reported, but not benchmarked. Here we compared urea-with AGPC-based protein extraction, profiling phosphorylations in the DNA damage response pathway after ionizing irradiation of U2OS cells as proof of principle. On average we identified circa 9000 phosphosites per sample with both extraction methods. Moreover, we observed high similarity of phosphosite characteristics (e.g. 94% shared class 1 identifications) and deduced kinase activities (e.g. ATM, ATR, CHEK1/2, PRKDC). AGPC-based sample extraction can thus replace standard cell lysates for phosphoproteomic workflows and may thus be an attractive way to obtain input material for multiple omics workflows, yielding several data types from a single sample.

scholarly journals An Optimised TRIzol-based Protocol for the Improvement of RNA Extraction Yield of Tomato Stem

2021 ◽  
Vol 44 (3) ◽  
Author(s):  
Anis Afifah ◽  
Prachumporn Nounurai ◽  
Rejeki Siti Ferniah ◽  
Hermin Pancasakti Kusumaningrum ◽  
Dyah Wulandari ◽  
...  
Keyword(s):  
Rna Extraction ◽  
Extraction Yield ◽  
Economic Feasibility ◽  
Rt Pcr ◽  
Rna Integrity ◽  
Guanidinium Thiocyanate ◽  
Sample Amount ◽  
Polymerase Chain ◽  
Yield And Purity

One of the most common methods for purifying RNA is using TRIzol reagent because of its simplicity and economic feasibility. However, the drawback of this method is frequently the low quality of extracted RNA due to contaminants from the residue of phenol and guanidinium thiocyanate from the reagents. This study aimed to evaluate the improvement in the quality and concentration of RNA after the optimisation treatment. One-month-old tomato (Solanum lycopersicum) stem was used in this research. TRIzol or acid guanidinium thiocyanate-phenol-chloroform-based method was given optimisation treatments of the initial sample amount, twice chloroform extraction, overnight precipitation at low temperature, and three times final washing with ethanol. The results showed no significant improvement (p > 0.05) in the purity ratio A260/A280. At the same time, there was a significant improvement (p < 0.05) in RNA yield and purity ratio A260/A230. The quality of RNA was verified using agarose-formaldehyde electrophoresis gel. Eight of nine samples (89%) from the optimised group had better RNA integrity characterised by sharp bands for 28S and 18S rRNA. Furthermore, a representative sample from the optimised group was successfully synthesised into complementary DNA by reverse transcriptase-polymerase chain reaction (RT-PCR) with primers of the ubiquitin (UBI3) gene. To sum up, optimised TRIzol-based protocol provides meaningful insight to produce RNA with better quality and suitability for downstream applications.

Comparative Study of Highly Pathogenic Avian Infl uenza Strains Isolated in Ukraine in 2005 and 2008

2014 ◽  
Vol 1 (1) ◽  
pp. 68-71
Author(s):  
A. Gerilovych ◽  
B. Stegniy ◽  
A. Stegniy ◽  
M. Stegniy ◽  
K. Smietanka ◽  
...  
Keyword(s):  
Western Europe ◽  
Rna Extraction ◽  
Experimental Studies ◽  
Molecular Characteristics ◽  
Eastern European ◽  
Highly Pathogenic ◽  
Genetic Lineages ◽  
Phylogenetic Studies ◽  
Polymerase Chain ◽  
Pathogenic H5n1

Objective. To research the molecular characteristics of two HPAI strains – A/Ch/Syvash/02/05/H5N1 and A/Ch/Krasnogvardeysk/58/08/H5N1, which were identifi ed as representatives of the highly pathogenic H5N1 viruses. Methods. RNA extraction, real-time polymerase chain reaction (PCR). Results. The phylogenetic studies revealed that the above mentioned strains belong to two various genetic lineages originated from the Eastern European strains isolated in 2005, but differ from the viruses introduced to the Central and Western Europe in 2005/2006, and also the lineages consisting of H5N1 viruses isolated in the Europe and Middle East in late 2007. Conclusions. Relying on experimental studies, it can be concluded that the strains of A/Ch/Syvash/02/05/H5N1 and A/Ch/Krasnogvardeysk/58/08/H5N1 are highly pathogenic.

Down regulation of COX-2, IL-1β, TNF-α in cynoviocyte by essential oil of Fraxinus excelsior

2018 ◽  
Vol 9 (03) ◽  
pp. 20192-20203
Author(s):  
Dr Maghsoudi, Hossein ◽  
Samaneh Haj-allahyari
Keyword(s):  
Liver Toxicity ◽  
Rna Extraction ◽  
Current Treatment ◽  
Fraxinus Excelsior ◽  
Peptic Ulcers ◽  
Down Regulation ◽  
Bovine Articular Cartilage ◽  
Tnf Α ◽  
Cytokine Levels ◽  
Cox 2

Osteoarthritis (arthritis) is biomechanical, biochemical and cellular phenomenon, and is not known as a degenerative disease. Arthritis is one of the common chronic diseases and the most important reason of physical disability in the world. According to its side effect such as peptic ulcers, gastrointestinal bleeding, liver toxicity and renal complications dueofprescribing current treatment contain corticosteroid and non-steroidal, we decided to evaluate possible effect of anti-inflammatory Esential oil of Fraxinus excelsior (EOFE) on biomarkers involved in disease. EOFE were prepared of genetic resources center. Bovine  articular cartilage derived from the metacarpophalangeal joints of 14–18-month-old animals (without any sign of inflammation and bleeding) sent to laboratory in sterile bags at 4ºC. Cells were cultured in appropriate condition and counted by hemocytometer, viability assessed by trypan blue. After LPS treatment, cytokine levels were assayed. Cells cultures again and were kept in 37C, 90% humidity in CO2 incubator and after RNA extraction, RT-PCR and PCR done. Also by Real-time PCR, gene expression was evaluated. E.E.F.E level cause down regulation of COX-2, IL-1β, TNF-α in LPS-stimulated cells.

Comparison of Different Methods for RNA Extraction from Capparis spinosa

2010 ◽  
Vol 26 (3) ◽  
pp. 401-405
Author(s):  
Dong-dong LUAN ◽  
Qing-hua SHI ◽  
Hong CHEN ◽  
Yu-zhi DONG
Keyword(s):  
Rna Extraction ◽  
Capparis Spinosa

The Frequency of HIV-1 Infection in Iranian Children and Determination of the Transmitted Drug Resistance in Treatment-Naïve Children

2020 ◽  
Vol 17 (6) ◽  
pp. 397-407
Author(s):  
Maryam Jarchi ◽  
Farah Bokharaei-Salim ◽  
Maryam Esghaei ◽  
Seyed Jalal Kiani ◽  
Fatemeh Jahanbakhsh ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Pediatric Patients ◽  
Phylogenetic Analyses ◽  
Rna Extraction ◽  
Transmitted Drug Resistance ◽  
The Arts ◽  
Treatment Naïve ◽  
Iranian Children ◽  
Hiv 1

Background: The advent of resistance-associated mutations in HIV-1 is a barrier to the success of the ARTs. Objective: In this study, the abundance of HIV-1 infection in Iranian children, and also detection of the TDR in naïve HIV-1 infected pediatric (under 12 years old) were evaluated. Materials: From June 2014 to January 2019, a total of 544 consecutive treatment-naïve HIV-1- infected individuals enrolled in this study. After RNA extraction, amplification, and sequencing of the HIV-1 pol gene, the DRM and phylogenetic analysis were successfully performed on the plasma specimens of the ART-naïve HIV-1-infected-children under 12 years old. The DRMs were recognized using the Stanford HIV Drug Resistance Database. Results: Out of the 544 evaluated treatment-naïve HIV-1-infected individuals, 15 (2.8%) cases were children under 12 years old. The phylogenetic analyses of the amplified region of pol gene indicated that all of the 15 HIV-1-infected pediatric patients were infected by CRF35_AD, and a total of 13.3% (2/15) of these children were infected with HIV-1 variants with SDRMs (one child harbored two related SDRMs [D67N, V179F], and another child had three related SDRMs [M184V, T215F, and K103N]), according to the last algorithm of the WHO. No PIs-related SDRMs were observed in HIV-1-infected children. Conclusion: The current study demonstrated that a total of 13.3% of treatment-naïve HIV-1-infected Iranian pediatrics (under 12 years old) were infected with HIV-1 variants with SDRMs. Therefore, it seems that screening to recognize resistance-associated mutations before the initiation of ARTs among Iranian children is essential for favorable medication efficacy and dependable prognosis.

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