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 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.

Loop-mediated Isothermal Amplification for Rapid Detection of Mating Types of Villosiclava virens

Plant Disease ◽  
2021 ◽  
Author(s):  
Xiayan Pan ◽  
Xiao Wang ◽  
Junjie Yu ◽  
Mina Yu ◽  
Huijuan Cao ◽  
...  
Keyword(s):  
Mating Type ◽  
Genomic Dna ◽  
High Efficiency ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Loop Mediated Isothermal Amplification ◽  
Villosiclava Virens ◽  
False Smut ◽  
Mating Type Genes ◽  
Polymerase Chain

Rice false smut (RFS), caused by Villosiclava virens, is an important fungal disease in panicle of rice. V. virens is a heterothallic ascomycete that controlled by two opposite idiomorphs, MAT1-1 and MAT1-2. Previous study showed sexual reproduction of V. virens plays an important role in the epidemic of RFS. In this study, we have developed a loop-mediated isothermal amplification (LAMP) assay to detect mating type of V. virens easily and rapidly by using specific primers designed on the mating type genes MAT1-1-2 and MAT1-2-1, respectively. The LAMP assay required only a water/dry bath and could recognize the mating type of V. virens in just 45 min. The LAMP assay was so sensitive that could detect small amounts of V. virens genomic DNA (as low as 2.0 pg of MAT1-1, and 200.0 pg of MAT1-2), which was 10-fold more sensitive than polymerase chain reaction (PCR). In addition, the application of mating type using LAMP assay was demonstrated feasibly by assessing the genomic DNA of V. virens isolated from rice fields. The high efficiency and specificity of this LAMP assay suggested it can be used as a rapid testing tool in mating type recognition of V. virens isolates in the field.

scholarly journals Inactivation of SARS-CoV-2 virus in saliva using a guanidium based transport medium suitable for RT-PCR diagnostic assays

PLoS ONE ◽  
2021 ◽  
Vol 16 (6) ◽  
pp. e0252687
Author(s):  
Sukalyani Banik ◽  
Kaheerman Saibire ◽  
Shraddha Suryavanshi ◽  
Glenn Johns ◽  
Soumitesh Chakravorty ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Viral Rna ◽  
Clinical Samples ◽  
Sample Collection ◽  
Rt Pcr ◽  
Diagnostic Assays ◽  
Guanidinium Thiocyanate ◽  
Upper Respiratory ◽  
Polymerase Chain ◽  
The Stability

Background Upper respiratory samples used to test for SARS-CoV-2 virus may be infectious and present a hazard during transport and testing. A buffer with the ability to inactivate SARS-CoV-2 at the time of sample collection could simplify and expand testing for COVID-19 to non-conventional settings. Methods We evaluated a guanidium thiocyanate-based buffer, eNAT™ (Copan) as a possible transport and inactivation medium for downstream Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) testing to detect SARS-CoV-2. Inactivation of SARS-CoV-2 USA-WA1/2020 in eNAT and in diluted saliva was studied at different incubation times. The stability of viral RNA in eNAT was also evaluated for up to 7 days at room temperature (28°C), refrigerated conditions (4°C) and at 35°C. Results SARS-COV-2 virus spiked directly in eNAT could be inactivated at >5.6 log10 PFU/ml within a minute of incubation. When saliva was diluted 1:1 in eNAT, no cytopathic effect (CPE) on VeroE6 cells was observed, although SARS-CoV-2 RNA could be detected even after 30 min incubation and after two cell culture passages. A 1:2 (saliva:eNAT) dilution abrogated both CPE and detectable viral RNA after as little as 5 min incubation in eNAT. SARS-CoV-2 RNA from virus spiked at 5X the limit of detection remained positive up to 7 days of incubation in all tested conditions. Conclusion eNAT and similar guanidinium thiocyanate-based media may be of value for transport, stabilization, and processing of clinical samples for RT-PCR based SARS-CoV-2 detection.

scholarly journals Monitoring of rotavirus in treated wastewater in Tehran with a monthly interval, in 2017–2018

2020 ◽  
Vol 18 (6) ◽  
pp. 1065-1072
Author(s):  
Shadi Tavakoli Nick ◽  
Seyed Reza Mohebbi ◽  
Seyed Masoud Hosseini ◽  
Hamed Mirjalali ◽  
Masoud Alebouyeh
Keyword(s):  
Rna Extraction ◽  
Conventional Polymerase Chain Reaction ◽  
Treated Wastewater ◽  
Harsh Environments ◽  
Monthly Interval ◽  
Rt Pcr ◽  
Conventional Pcr ◽  
Wastewater Treatment Process ◽  
Polymerase Chain ◽  
Elution Method

Abstract Rotaviruses are among the major causes of viral acute gastroenteritis in newborns and children younger than 5 years worldwide. The ability of rotaviruses to remain infectious in harsh environments as well as in the wastewater treatment process makes them one of the most prevalent enteric viruses. The current study aimed to determine the presence of rotavirus genomes and to analyze them phylogenetically in secondary treated wastewater (TW) samples. In total, 13 TW samples were collected from September 2017 to August 2018. Viral concentration was carried out using the absorption-elution method, and after RNA extraction and cDNA synthesis, real-time and conventional polymerase chain reaction (PCR) were performed. A phylogenetic tree was drawn using Maximum Likelihood and Tamura 3-parameter using MEGA v.6 software. Rotavirus genomes were detected in 7/13 (53.8%) and 3/13 (23.07%) samples using reverse transcription (RT)-PCR and conventional PCR, respectively. Accordingly, phylogenetic analysis revealed G4P[8], G9P[4], and G9P[8] genotypes among the samples. The presence of rotavirus in secondary TW samples discharged into surface water emphasizes the importance of monitoring and assessing viral contamination in the water sources used for agricultural and recreational purposes.

scholarly journals Evaluation of COVID-19 RT-qPCR Test in Multi sample Pools

2020 ◽  
Vol 71 (16) ◽  
pp. 2073-2078 ◽  
Author(s):  
Idan Yelin ◽  
Noga Aharony ◽  
Einat Shaer Tamar ◽  
Amir Argoetti ◽  
Esther Messer ◽  
...  
Keyword(s):  
Quantitative Polymerase Chain Reaction ◽  
False Negative ◽  
Rna Extraction ◽  
False Negative Rate ◽  
Positive Sample ◽  
Recent Emergence ◽  
Single Positive ◽  
Polymerase Chain ◽  
Pool Test ◽  
Current Screening

Abstract Background The recent emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) led to a current pandemic of unprecedented scale. Although diagnostic tests are fundamental to the ability to detect and respond, overwhelmed healthcare systems are already experiencing shortages of reagents associated with this test, calling for a lean immediately applicable protocol. Methods RNA extracts of positive samples were tested for the presence of SARS-CoV-2 using reverse transcription quantitative polymerase chain reaction, alone or in pools of different sizes (2-, 4-, 8-, 16-, 32-, and 64-sample pools) with negative samples. Transport media of additional 3 positive samples were also tested when mixed with transport media of negative samples in pools of 8. Results A single positive sample can be detected in pools of up to 32 samples, using the standard kits and protocols, with an estimated false negative rate of 10%. Detection of positive samples diluted in even up to 64 samples may also be attainable, although this may require additional amplification cycles. Single positive samples can be detected when pooling either after or prior to RNA extraction. Conclusions As it uses the standard protocols, reagents, and equipment, this pooling method can be applied immediately in current clinical testing laboratories. We hope that such implementation of a pool test for coronavirus disease 2019 would allow expanding current screening capacities, thereby enabling the expansion of detection in the community, as well as in close organic groups, such as hospital departments, army units, or factory shifts.

Investigation of GSTP1 and epigenetic regulators expression pattern in a population of Iranian patients with prostate cancer

2020 ◽  
Vol 28 (4) ◽  
pp. 327-334
Author(s):  
Mahan Mohammadi ◽  
Shiva Irani ◽  
Iman Salahshourifar ◽  
Jalil Hosseini ◽  
Afshin Moradi ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
High Efficiency ◽  
Rna Extraction ◽  
P Value ◽  
Prostate Hyperplasia ◽  
Expression Of Genes ◽  
Elevated Expression ◽  
Epigenetic Regulators ◽  
The Impact ◽  
Iranian Patients

BACKGROUND AND AIM: Prostate cancer is the leading cause of death in many countries. It is important to diagnose the disease in the early stages. Current methods detect the disease with low specificity. Examining the expression of genes responsible for disease and their epigenetic regulators are good tools in this regard. MATERIAL AND METHODS: In this prospective case-control study, 40 Iranian patients with cancer, 40 Iranian patients with prostate hyperplasia, and 40 control samples were examined. After blood sampling from each individual, RNA extraction and cDNA synthesis, GSTP1, HDAC, DNMT3A, and DNMT3B expressions were measured in three understudy groups using specific primers and Real-Time PCR method. RESULTS: A reverse correlation was identified between loss of GSTP1 expression and overexpression of HDAC, DNMT3A, and DNMT3B (P value < 0.0001) with a beneficial pattern of cancer development with high efficiency. The significant decrease of GSTP1 expression in patients in comparison to the healthy controls and the elevated expression levels of the studied epigenetic regulators in PCA and BPH samples indicate the impact of the regulators on GSTP1 expression activity. CONCLUSION: This study showed that the measurement of combined GSTP1 and its epigenetic regulators’ expression could be used as suitable genetic markers for the detection and separation of healthy individuals from prostatic patient groups in the Iranian population. However, a similar study in a larger population of case and control could help us to distinguish between normal, benign, and malignant conditions.

scholarly journals High Throughput T Epitope Mapping and Vaccine Development

2010 ◽  
Vol 2010 ◽  
pp. 1-12 ◽  
Author(s):  
Giuseppina Li Pira ◽  
Federico Ivaldi ◽  
Paolo Moretti ◽  
Fabrizio Manca
Keyword(s):  
T Cell ◽  
High Throughput ◽  
Epitope Mapping ◽  
Vaccine Development ◽  
Human Subjects ◽  
T Cell Response ◽  
Limiting Factor ◽  
T Cell Epitopes ◽  
Large Panels ◽  
Cell Assays

Mapping of antigenic peptide sequences from proteins of relevant pathogens recognized by T helper (Th) and by cytolytic T lymphocytes (CTL) is crucial for vaccine development. In fact, mapping of T-cell epitopes provides useful information for the design of peptide-based vaccines and of peptide libraries to monitor specific cellular immunity in protected individuals, patients and vaccinees. Nevertheless, epitope mapping is a challenging task. In fact, large panels of overlapping peptides need to be tested with lymphocytes to identify the sequences that induce a T-cell response. Since numerous peptide panels from antigenic proteins are to be screened, lymphocytes available from human subjects are a limiting factor. To overcome this limitation, high throughput (HTP) approaches based on miniaturization and automation of T-cell assays are needed. Here we consider the most recent applications of the HTP approach to T epitope mapping. The alternative or complementary use of in silico prediction and experimental epitope definition is discussed in the context of the recent literature. The currently used methods are described with special reference to the possibility of applying the HTP concept to make epitope mapping an easier procedure in terms of time, workload, reagents, cells and overall cost.

Thermal Analysis of a Micro-Polymerase Chain Reaction Device

2004 ◽  
Author(s):  
Jeff Punch ◽  
Bryan Rodgers ◽  
David Newport ◽  
Mark Davies
Keyword(s):  
Thermal Analysis ◽  
Polymerase Chain Reaction ◽  
High Efficiency ◽  
Closed Form Solution ◽  
Thermal Control ◽  
Rate Of Change ◽  
Temperature Cycling ◽  
Chain Reaction ◽  
Macro Scale ◽  
Polymerase Chain

Micro-scale polymerase chain reaction (micro-PCR) systems offer substantial advantages over macro-scale systems. Smaller sample volumes are required, and faster process times are feasible. Thermal control of micro-PCR systems is a substantial technical challenge, however. The PCR process requires the fluid sample to be cycled through three temperature ranges — typically 90–95°C, 50–65°C and 72–77°C for denaturation, hybridisation and replication respectively. Durations of the three steps are required to be in the ratio of 4:4:9. In this paper, the thermal analysis of a continuous flow micro-PCR device is reported. The objective of the analysis is to optimize the thermal performance of the device for fast amplification cycles with high efficiency - an efficient PCR features rapid heating and cooling between steps, and good temperature uniformity within each step. The device comprises an array of parallel microchannels formed within a polypropylene substrate to carry fluid, with the base of the substrate mounted on an aluminium carrier. Substrate depth is 500 micron, and each channel is 60 micron wide by 40 micron deep. Thermoelectric cells (TECs) are bonded to the carrier, and powered by a thermoelectric controller with feedback from sensors embedded in the carrier. A Pyrex Glass slide is bonded to the substrate to form closed channels. Arrays of film heaters mounted on the slide adjacent to the channel are used to establish the required temperature regions along the channel. By pumping the fluid at a fixed flow rate, temperature cycling of specific period is achieved. Thermal analysis of the substrate is performed using an approximate closed-form solution, in conjunction with Finite Element (FE) and Computational Fluid Dynamics (CFD) simulations. The analysis is used to conduct a parametric study in order to determine the optimum configurations of substrate materials, cooling conditions, heaters and flow rates required to impose specific temperature cycles. The use of thermoelectric cells is shown to increase the rate of change of temperature between the various regions, improving the efficiency and decreasing the cycle time of the PCR process. Cycle times of 6s or less are shown to be feasible, yielding benefits in time saved for multiple amplifications. Finally, the analysis is also used to identify the dimensionless parameters which govern the thermal characteristics of the device, illustrating the importance of the Biot number.

scholarly journals High efficiency photomodulators for millimeter wave and THz radiation

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
I. R. Hooper ◽  
N. E. Grant ◽  
L. E. Barr ◽  
S. M. Hornett ◽  
J. D. Murphy ◽  
...  
Keyword(s):  
High Efficiency ◽  
Continuous Wave ◽  
Broad Band ◽  
Cost Effective ◽  
Silicon Wafers ◽  
Thz Radiation ◽  
Limiting Factor ◽  
Switching Speed ◽  
Carrier Lifetimes ◽  
Narrow Frequency Band

AbstractPhotomodulators for mm-wave and THz radiation are an essential component for many imaging and signal processing applications. While a myriad of schemes have been devised to enhance photomodulation by enhancing the light-matter interaction, there has been less focus on the photoconductive materials themselves, which are often the limiting factor. Here, we present an approach to increase the photomodulation efficiency of silicon by orders of magnitude, using post treatment of off-the-shelf silicon wafers. The increase in efficiency removes the need for bulky and costly amplified laser sources, and creates the potential for compact and cost-effective modulators for real-world applications. By passivating the surfaces of long bulk-lifetime silicon wafers with Al2O3, the recombination of the photoexcited carriers at the surfaces is mostly eliminated. This results in vastly longer excess carrier lifetimes (up to ~50 ms), with corresponding increases in photoconductivity. The resulting modulators are highly efficient, with the transmission through them being reduced from ~90% to <10% over a narrow frequency band with a continuous wave excitation intensity of just 10 Wm−2, whilst modulation factors of greater than 80% can be achieved over a broad band with similar intensities. We also discuss the limitations of such long-lifetime modulators for applications where the switching speed or spatial resolution of a modulator may be critical.

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