rna extraction
Recently Published Documents


TOTAL DOCUMENTS

1654
(FIVE YEARS 842)

H-INDEX

51
(FIVE YEARS 14)

2022 ◽  
Vol 8 ◽  
Author(s):  
Paul Hofman ◽  
Olivier Bordone ◽  
Emmanuel Chamorey ◽  
Jonathan Benzaquen ◽  
Renaud Schiappa ◽  
...  

Introduction: Aside from the reverse transcription-PCR tests for the diagnosis of the COVID-19 in routine clinical care and population-scale screening, there is an urgent need to increase the number and the efficiency for full viral genome sequencing to detect the variants of SARS-CoV-2. SARS-CoV-2 variants assessment should be easily, rapidly, and routinely available in any academic hospital.Materials and Methods: SARS-CoV-2 full genome sequencing was performed retrospectively in a single laboratory (LPCE, Louis Pasteur Hospital, Nice, France) in 103 SARS-CoV-2 positive individuals. An automated workflow used the Ion Ampliseq SARS-CoV-2 panel on the Genexus Sequencer. The analyses were made from nasopharyngeal swab (NSP) (n = 64) and/or saliva (n = 39) samples. All samples were collected in the metropolitan area of the Nice city (France) from September 2020 to March 2021.Results: The mean turnaround time between RNA extraction and result reports was 30 h for each run of 15 samples. A strong correlation was noted for the results obtained between NSP and saliva paired samples, regardless of low viral load and high (>28) Ct values. After repeated sequencing runs, complete failure of obtaining a valid sequencing result was observed in 4% of samples. Besides the European strain (B.1.160), various variants were identified, including one variant of concern (B.1.1.7), and different variants under monitoring.Discussion: Our data highlight the current feasibility of developing the SARS-CoV-2 next-generation sequencing approach in a single hospital center. Moreover, these data showed that using the Ion Ampliseq SARS-CoV-2 Assay, the SARS-CoV-2 genome sequencing is rapid and efficient not only in NSP but also in saliva samples with a low viral load. The advantages and limitations of this setup are discussed.


2022 ◽  
pp. 16-21
Author(s):  
Baraa Akeel Al-Hasan ◽  
Abdullah O. Alhatami ◽  
Husam Muhsen Abdulwahab ◽  
Ghadeer Sabah Bustani ◽  
Muhammad Ali Hameed ◽  
...  

Background and Aim: Swollen head syndrome (SHS) is a complex disease caused by various agents, including bacterial and viral pathogens, as well as environmental factors. Avian metapneumovirus (aMPV) is one of the most important causes of respiratory diseases and SHS in poultry and one of the most widespread viruses worldwide; however, it has not been recorded in Iraq. This study aimed at the molecular identification and subtyping of aMPV in poultry, with the objectives of investigating the prevalence of aMPV in infected broiler flocks with SHS and molecular typing using primers specific to the study of the prevalence of subtypes A, B, and C of aMPV. Materials and Methods: This study was performed on 67 broiler farms that reported typical SHS from September 2018 to August 2019. Swabs were collected from the trachea, infraorbital sinuses, and lung, then uploaded on FTA cards and subjected to an RNA extraction protocol. Results: aMPV was detected in 16 (23.8%) samples. Molecular typing using primers specific to the attachment glycoprotein (G) gene showed that all positive samples belonged to subtype B, as assessed using the real-time polymerase chain reaction technique. Conclusion: aMPV may be the main etiological factor causing SHS in poultry. Moreover, this was the first report of the prevalence of subtype B aMPV strains in broiler farms in Iraq.


2022 ◽  
Author(s):  
Bongkot Ngamsom ◽  
Alexander Iles ◽  
Moses Kamita ◽  
Racheal Kimani ◽  
Pablo Rodriguez-Mateos ◽  
...  

In response to the ongoing COVID-19 pandemic and disparities of vaccination coverage in low-and middle-income countries, it is vital to adopt a widespread testing and screening programme, combined with contact tracing, to monitor and effectively control the infection dispersion in areas where medical resources are limited. This work presents a lab-on-a-chip platform, namely IFAST-CRISPR, as an affordable, rapid and high-precision molecular diagnostic means for SARS-CoV-2 detection. The herein proposed sample-to-answer platform integrates RNA extraction, amplification and CRISPR-Cas-based detection with lateral flow readout in one device. The microscale dimensions of the device containing immiscible liquids, coupled with the use of silica paramagnetic beads and GuHCl, streamline sample preparation, including RNA concentration, extraction and purification, in 15 min with minimal hands-on steps. By combining RT-LAMP with CRISPR-Cas12 assays targeting the nucleoprotein (N) gene, visual identification of ≥ 470 copies mL-1 genomic SARS-CoV-2 samples was achieved in 45 min, with no cross-reactivity towards HCoV-OC43 nor H1N1. On-chip assays showed the ability to isolate and detect SARS-CoV-2 from 1,000 genome copies mL-1 of replication-deficient viral particles in 1 h. This simple, affordable and integrated platform demonstrated a visual, faster, and yet specificity and sensitivity-comparable alternative to the costly gold-standard RT-PCR assay, requiring only a simple heating source. Further investigations on multiplexing and direct interfacing of the accessible Swan-brand cigarette filter for saliva sample collection could provide a complete work flow for COVID-19 diagnostics from saliva samples suitable for low-resource settings.


2022 ◽  
Author(s):  
Joost S S. Mansour ◽  
Konstantinos Anestis ◽  
Fabrice Not ◽  
Uwe John

Many marine protists are not culturable and therefore challenging to study, nonetheless, they are essential in all marine ecosystems. The development of single-cell techniques is allowing for more marine protists to be studied. Such genomic approaches aim to help to disentangle heterotrophic processes such as phagotrophy from osmotrophy and phototrophic-induced anabolic activities. This information will then support cellular and metabolic modeling by better elucidating the physiological mechanisms and quantifying their importance in different scenarios. However, single-cell protocols and low input RNA kits for transcriptomics are usually made for and tested with mammalian cells, as such the feasibility and efficiency of single-cell transcriptomics on highly diverse mixotrophic protists is not always known. Often single-cell transcriptomics of microbial eukaryotes shows low transcript recovery rates and large variability. We report on transcriptomic methods that we have successfully performed on single cells of Acantharia, Strombidium basimorphum, and Prymnesium parvum. This protocol follows up after total RNA extraction (from the protocol at dx.doi.org/10.17504/protocols.io.bp6xmrfn) to prepare cDNA libraries for Illumina sequencing. The described protocol uses the SMART-Seq4 kit (Takara #634891) for cDNA synthesis and amplification, but this can also be successfully performed with the NEBNext kit (NEB #E6421). The NEBNext kit protocol is very similar to the protocol described here and generally the manufacture's protocol can be followed but see the notes at step 4 and step 18 of this protocol, and do the final elution after cDNA purification in 10 mM Tris (pH 8.0). The subsequent cDNA library is prepared following the .


Author(s):  
Priscilla Gomes da Silva ◽  
José Gonçalves ◽  
Ariana Isabel Brito Lopes ◽  
Nury Alves Esteves ◽  
Gustavo Emanuel Enes Bamba ◽  
...  

As the third wave of the COVID-19 pandemic hit Portugal, it forced the country to reintroduce lockdown measures due to hospitals reaching their full capacities. Under these circumstances, environmental contamination by SARS-CoV-2 in different areas of one of Portugal’s major Hospitals was assessed between 21 January and 11 February 2021. Air samples (n = 44) were collected from eleven different areas of the Hospital (four COVID-19 and seven non-COVID-19 areas) using Coriolis® μ and Coriolis® Compact cyclone air sampling devices. Surface sampling was also performed (n = 17) on four areas (one COVID-19 and three non-COVID-19 areas). RNA extraction followed by a one-step RT-qPCR adapted for quantitative purposes were performed. Of the 44 air samples, two were positive for SARS-CoV-2 RNA (6575 copies/m3 and 6662.5 copies/m3, respectively). Of the 17 surface samples, three were positive for SARS-CoV-2 RNA (200.6 copies/cm2, 179.2 copies/cm2, and 201.7 copies/cm2, respectively). SARS-CoV-2 environmental contamination was found both in air and on surfaces in both COVID-19 and non-COVID-19 areas. Moreover, our results suggest that longer collection sessions are needed to detect point contaminations. This reinforces the need to remain cautious at all times, not only when in close contact with infected individuals. Hand hygiene and other standard transmission-prevention guidelines should be continuously followed to avoid nosocomial COVID-19.


2022 ◽  
Vol 22 (1) ◽  
Author(s):  
Mohammad E. Khamseh ◽  
Alireza Sheikhi ◽  
Zahra Shahsavari ◽  
Mohammad Ghorbani ◽  
Hamideh Akbari ◽  
...  

Abstract Background Pituitary adenomas impose a burden of morbidity on patients and characterizing the molecular mechanisms underlying its pathogenesis received remarkable attention. Despite the appealing role of necroptosis as an alternative cell death pathway in cancer pathogenesis, its relevance to pituitary adenoma pathogenesis has yet to be determined that is perused in the current study. Methods The total number of 109 specimens including pituitary adenomas and cadaveric healthy pituitary tissues were enrolled in the current study. Tumor and healthy pituitary tissues were subjected to RNA extraction and gene analysis using Real-Time PCR. The expression levels of necroptosis markers (RIP1K, RIP3K and, MLKL) and their association with the patient’s demographic features were evaluated, also the protein level of MLKL was assessed using immunohistochemistry in tissues. Results Based on our data, the remarkable reduction in RIP3K and MLKL expression were detected in nonfunctional and GH-secreting pituitary tumors compared to pituitary normal tissues. Invasive tumors revealed lower expression of RIP3K and MLKL compared to non-invasive tumors, also the attenuated level of MLKL was associated with the tumor size in invasive NFPA. The simultaneous down-regulation of MLKL protein in pituitary adenoma tissues was observed which was in line with its gene expression. While, RIP1K over-expressed significantly in both types of pituitary tumors which showed no significant correlation with patient’s age, gender and tumor size in GHPPA and NFPA group. Notably, MLKL and RIP3K gene expression was significantly correlated in the GHPPA group. Conclusions According to our data, the reduced expression of necroptosis mediators (RIP3K, MLKL) in pituitary adenoma reinforces the hypothesis that the necroptosis pathway can be effective in regulating the proliferation and growth of pituitary tumor cells and tumor recurrence.


2022 ◽  
Vol 9 ◽  
Author(s):  
Itay Bar-Or ◽  
Karin Yaniv ◽  
Marilou Shagan ◽  
Eden Ozer ◽  
Merav Weil ◽  
...  

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an RNA virus, a member of the coronavirus family of respiratory viruses that includes severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) and the Middle East respiratory syndrome (MERS). It has had an acute and dramatic impact on health care systems, economies, and societies of affected countries during the past 8 months. Widespread testing and tracing efforts are being employed in many countries in attempts to contain and mitigate this pandemic. Recent data has indicated that fecal shedding of SARS-CoV-2 is common and that the virus RNA can be detected in wastewater. This indicates that wastewater monitoring may provide a potentially efficient tool for the epidemiological surveillance of SARS-CoV-2 infection in large populations at relevant scales. In particular, this provides important means of (i) estimating the extent of outbreaks and their spatial distributions, based primarily on in-sewer measurements, (ii) managing the early-warning system quantitatively and efficiently, and (iii) verifying disease elimination. Here we report different virus concentration methods using polyethylene glycol (PEG), alum, or filtration techniques as well as different RNA extraction methodologies, providing important insights regarding the detection of SARS-CoV-2 RNA in sewage. Virus RNA particles were detected in wastewater in several geographic locations in Israel. In addition, a correlation of virus RNA concentration to morbidity was detected in Bnei-Barak city during April 2020. This study presents a proof of concept for the use of direct raw sewage-associated virus data, during the pandemic in the country as a potential epidemiological tool.


2022 ◽  
Vol 21 (10) ◽  
Author(s):  
Mitra Nasiri ◽  
Zohreh Yousefi Ghalejoogh ◽  
Angila Ataei-Pirkooh ◽  
Farah Bokharaei-Salim ◽  
Seyed Hamidreza Monavari ◽  
...  

Background: Limited sources of fresh water necessitate the application of health policies for treatment and decontamination of human sewage for further use. A wide variety of infectious agents, including bacteria, fungi, parasites, and viruses, can be found in sewage. Enteric viruses such as hepatitis A virus (HAV) can survive the current treatments and infect susceptible hosts. Objectives: This study aimed to evaluate the HAV contamination in human sewage before and after treatment in the wastewater treatment plant of Ekbatan town in Tehran, Iran, and analyze the phylogenetic properties of the identified viruses. Methods: Over a 12-month period, we collected the wastewater samples including influent, before chlorination, and effluent, from the wastewater treatment plant of Ekbatan town in Tehran, Iran. Ribonucleic acid (RNA) extraction, complementary deoxyribonucleic acid (cDNA) synthesis, and semi-nested polymerase chain reaction (PCR) were performed to identify HAV contamination. Phylogenetic analysis was performed to investigate subgenotypes of the virus. Results: HAV was detected in all influents and samples before chlorination, while the virus was detected in 50% of the effluent samples. All detected viruses belonged to subgenotype IB. Conclusions: Investigating the presence of HAV in sewage provides a general picture of the virus spread in the population of interest. HAV was detected in all influent samples, indicating that the infection is endemic in this area all year round. This also indicates the inability of the current treatment protocols in virus removal, which can be a threat to the public health.


2022 ◽  
Vol 67 (4) ◽  
pp. 321-327
Author(s):  
Jiang Lin ◽  
Qi Ding ◽  
Guoying Zhang ◽  
Xiling Yin

Breast cancer is the most common cancer among women in the world. The phosphatidylinositol 3-Kinase (PI3k), which regulates various cellular signaling pathways, is often elevated in human cancers. This study aimed to evaluate the expression of the PI3k gene in breast cancer. In this case-control study, 40 paraffin-embedded tissues of breast cancer and 40 adjacent non-tumor tissues were examined. After total RNA extraction and cDNA synthesis, the relative expression of the gene was obtained using the real-time-PCR method and evaluated by the 2-ΔΔCT method. Also, the association of gene expression with clinical factors and survival rate was investigated. Data analysis was performed by SPSS statistical software (version 22), t-test, and ANOVA. A p-value of less than 0.05 was considered significant. The results showed that PI3k expression was significantly increased in breast tumor tissues compared to non-tumor tissues (p = 0001). Consistent with these results, PI3k expression was associated with metastasis (p = 0.008) and high tumor grade (p = 0.01). In addition, increasing PI3k expression decreased overall survival compared to its low expression (p = 0.03). In general, PI3k plays a tumor-enhancing role in the progression of breast cancer. In addition, increased PI3k expression is associated with metastasis and poor prognosis of cancer, so that PI3k may be useful in the diagnosis, treatment, and prognosis of people with the disease. However, further investigation is needed to substantiate this claim.


2022 ◽  
Vol 67 (4) ◽  
pp. 367-375
Author(s):  
Jalal Hamasalih Fattah

Follicular unit extraction (FUE) has evolved dramatically as the most recent advancement in surgical hair restoration as it leaves a tiny scar and creates natural and pleasing results. This study aims to show the effectiveness of adjuvant measures and genetic evaluations in improving outcomes. Prospective analysis of 271 male patients with androgenic alopecia who underwent hair transplantation with FUE technique between August 2015 and February 2020 at our center was conducted. The mean age was 35.93 ±4.40 years. At one year postoperatively, patients were asked to fill up a questionnaire which included their satisfaction level, need for 2nd session, and complications. Informed written consent was obtained from all patients. Also, blood samples were provided from patients before the operation. RNA extraction and cDNA synthesis were performed using the RNX-Plus kit (Cinnagen, Iran) and Vivantis kit (Malaysia). Amplification of SRD5A2 and GAPDH genes (as internal standard) for measuring gene expression was performed by real-time PCR. Data were analyzed using the statistical package for social science SPSS V. 23. In the last 156 cases, the addition of 40 mg of Triamcinolone to the LA solution led to a dramatic reduction of the incidence of postoperative oedema, from 40% to 9%. Adding three sessions of PRP at 2nd, 4th and 6th months postoperatively resulted in an increased patient satisfaction rate with better hair density and thickness where the rate of highly satisfied patients increased from 64.5% to 83.7%. The addition of 40 mg Triamcinolone to the LA solution was highly effective in reducing postoperative oedema. Three sessions of PRP at 2nd, 4th and 6th months postoperatively were recommended. The results of SRD5A2 gene expression showed that the expression of this gene in satisfied (P = 0.049) and dissatisfied (P = 0.028) patients were significantly higher than highly satisfied patients, which means that the SRD5A2 gene expression had an essential role in the successfulness of hair transplantation. The increased expression of this gene could reduce the response to hair transplantation.


Sign in / Sign up

Export Citation Format

Share Document