Search results for "Reverse Transcription"
showing 10 items of 50 documents
Manganese Ions Individually Alter the Reverse Transcription Signature of Modified Ribonucleosides
2020
Reverse transcription of RNA templates containing modified ribonucleosides transfers modification-related information as misincorporations, arrest or nucleotide skipping events to the newly synthesized cDNA strand. The frequency and proportion of these events, merged from all sequenced cDNAs, yield a so-called RT signature, characteristic for the respective RNA modification and reverse transcriptase (RT). While known for DNA polymerases in so-called error-prone PCR, testing of four different RTs by replacing Mg2+ with Mn2+ in reaction buffer revealed the immense influence of manganese chloride on derived RT signatures, with arrest rates on m1A positions dropping from 82% down to 24%. Additi…
Machine learning of reverse transcription signatures of variegated polymerases allows mapping and discrimination of methylated purines in limited tra…
2020
AbstractReverse transcription (RT) of RNA templates containing RNA modifications leads to synthesis of cDNA containing information on the modification in the form of misincorporation, arrest, or nucleotide skipping events. A compilation of such events from multiple cDNAs represents an RT-signature that is typical for a given modification, but, as we show here, depends also on the reverse transcriptase enzyme. A comparison of 13 different enzymes revealed a range of RT-signatures, with individual enzymes exhibiting average arrest rates between 20 and 75%, as well as average misincorporation rates between 30 and 75% in the read-through cDNA. Using RT-signatures from individual enzymes to trai…
The reverse transcription signature of N-1-methyladenosine in RNA-Seq is sequence dependent
2015
The combination of Reverse Transcription (RT) and high-throughput sequencing has emerged as a powerful combination to detect modified nucleotides in RNA via analysis of either abortive RT-products or of the incorporation of mismatched dNTPs into cDNA. Here we simultaneously analyze both parameters in detail with respect to the occurrence of N-1-methyladenosine (m1A) in the template RNA. This naturally occurring modification is associated with structural effects, but it is also known as a mediator of antibiotic resistance in ribosomal RNA. In structural probing experiments with dimethylsulfate, m1A is routinely detected by RT-arrest. A specifically developed RNA-Seq protocol was tailored to …
Translocation (X;18) in a Biphasic Synovial Sarcoma with Morphologic Features of Neural Differentiation
1998
The authors report a recurred neoplasm showing distinctive histologic, immunophenotypic, and ultrastructural features characteristic of biphasic synovial sarcoma with neural differentiation. The features include areas with a growth pattern of densely packed spindle cells in irregularly intersecting, broad fascicles, diffuse vimentin and HBA 71 immunoreactivity, expression of S-100 protein, and other neural markers. Moreover, areas with glandular structures and cellular expression of cytokeratin and epithelial membrane antigen were noted. Additionally, areas of neural-like growth pattern were positive for neuron-specific enolase, HNK-1, and protein gene product 9.5. Furthermore, cytogenetic …
Cardiac surgery outcomes in patients with coronavirus disease 2019 (COVID-19): A case-series report
2020
Abstract Objective The impact of coronavirus disease 2019 (COVID-19) on the postoperative course of patients after cardiac surgery is unknown. We experienced a major severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak in our cardiac surgery unit, with several patients who tested positive early after surgery. Here we describe the characteristics, postoperative course, and laboratory findings of these patients, along with the fate of the health care workers. We also discuss how we reorganize and reallocate hospital resources to resume the surgical activity without further positive patients. Methods After diagnosis of the first symptomatic patient, surgery was suspended. Naso…
Detection of human immunodeficiency virus-1 DNA, RNA and antibody, and occult blood in inactivated saliva: Availability of the filter paper disk meth…
1996
Eighty three samples of saliva were collected from 60 subjects who were asymptomatic carriers of AIDS related complex (ARC) and AIDS. They included hemophiliacs, homosexuals and heterosexuals. Occult blood, human immunodeficiency virus-1 (HIV-1) antibody and DNA were assayed after heat treatment at 56 degrees C by strip method, particle agglutination and polymerase chain reaction (PCR), respectively. HIV-1 RNA was assayed by reverse transcription (RT)-PCR after heat treatment at 56 degrees C or 90 degrees C, or after application to filter paper disks with drying and heat treatment at 90 degrees C. Positive results were found in 53% (occult blood), 73% (HIV-1 antibody), 23% (HIV-1 DNA, 56 de…
Overexpression of the insulin-like growth factor I receptor in human pheochromocytomas.
2006
In order to determine the role of the IGF-I receptor (IGF-IR) in human pheochromocytomas we have compared the expression of the IGF-IR in normal tissues and in pheochromocytomas with regard to the IGF-IR mRNA levels and ligand binding. By semiquantitative reverse transcription polymerase chain reaction (RT-PCR), the mRNA of the IGF-IR could be detected in all samples of normal adrenomedullary cells (n=13) and pheochromocytomas (n=16). However, pheochromocytomas exhibited 2.8-fold higher mean IGF-IR mRNA levels than normal adrenomedullary cells (2.8±0.5×105 molecules/μg RNA vs 7.8±1.2×105 molecules/μg RNA; P < 0.001). This overexpression of the IGF-IR in pheochromocytomas could be confirm…
Evaluation of saliva as a complementary technique to the diagnosis of COVID-19:a systematic review
2021
Background Infectious disease coronavirus 2019 (COVID-19) is caused by the SARS-CoV-2 virus, and it mainly affects the upper respiratory tract. The gold standard for its diagnosis is real-time reverse transcription polymerase chain reaction (RT-qPCR) performed on a nasopharyngeal swab. In contrast, testing saliva has significant advantages as a diagnostic method. Material and Methods We searched for articles evaluating saliva as a diagnostic method for COVID-19 on the PUBMED/MEDLINE, WEB OF SCIENCE, COCHRANE, and SCIELO platforms. We initially found 233 articles and 20 were selected for inclusion following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses protocol: 18 c…
Expression of cytokeratin 17 mRNA in oral squamous cell carcinoma cells obtained by brush biopsy: preliminary results.
2009
Background: The aim of this study was to determine the detection of cytokeratin (CK) mRNA in oral squamous cell carcinoma (OSCC) cells and to evaluate the CK relevance for OSCC diagnosis in a brush biopsy test. Methods: Fifty-two pairs of OSCC cells and normal oral mucosal cells were obtained by brush biopsy from OSCC patients. mRNA was extracted from cell pellets for real-time quantitative reverse transcriptase polymerase chain reaction (RT-qPCR). The over-expression levels of CK 17, CK 19 and CK 20 mRNA in OSCC cells were examined by SYBR green real-time RT-qPCR. Results: Compared to normal mucosal cells, the over-expression of CK 17 mRNA was detectable in 40 OSCC cells (76.9%), that o…
Differential expression of Cryptosporidium parvum genes encoding sporozoite surface antigens in infected HCT-8 host cells.
2006
Intracellular replication of Cryptosporidium parvum (Apicomplexa) involves the generation of several asexual and sexual forms of the parasite. During the stage conversions, complex mechanisms lead to differential structural and functional properties of the parasite. These require a well tuned gene transcription machinery. For the first time the gene expression of four surface proteins of C. parvum sporozoites, CP15, CP17, P23, and GP900 were analysed in parallel by reverse transcription polymerase chain reaction. In addition, CP17 and P23 antigens were detected in infected host cells by immunofluorescence using antisera raised against recombinant forms of the proteins. The results show that…