scholarly journals Determination of m6A frequency utilizing 4SedTTP-RT Ligation Assisted PCR (SLAP) in viral and cellular long non-coding RNAs

2021 ◽  
Author(s):  
Sarah Elizabeth Martin ◽  
Huachen Gan ◽  
Joanna Sztuba-Solinska

N6-methyladenosine is one of the most abundant epitranscriptomic signatures that can affect every aspect of RNA biology, from structure and stability to intra- and intermolecular interactions. The accurate quantitative assessment of RNA stoichiometry at single-nucleotide resolution is a prerequisite to evaluate the biological significance of m6A in the context of specific RNA. We have developed a new method, termed 4-Selenothymidine 5′-triphosphate reverse transcription and Ligation Assisted PCR analysis (SLAP), for quantitative and unbiased assessment of the m6A fraction on target RNA. The inclusion of thymidine triphosphate derivative during reverse transcription discourages base pair formation with m6A resulting in the reaction's cessation, while maintaining normal A-T base pairing. The site-specific ligation of the resulting cDNAs with adapters, followed by amplification, generates two distinct products that reflect the modified and unmodified fraction of the analyzed RNA. These PCR products are subsequently separated by gel electrophoresis and quantified using densitometric analysis. We applied the SLAP to verify the position and assess the frequency of m6A sites present on two exemplary long non-coding RNAs. We assessed the SLAP specificity, accuracy, and sensitivity, proving the applicability of this method for the m6A analysis on less abundant transcripts. Overall, this method constitutes an extension of the bird's-eye view of RNA m6A landscape provided by epitranscriptome-wide analyses by delivering quantitative assessment of modification frequency and can therefore aid the understanding of the consequences of m6A on biological processes.

2019 ◽  
Author(s):  
Lucia Lorenzi ◽  
Hua-Sheng Chiu ◽  
Francisco Avila Cobos ◽  
Stephen Gross ◽  
Pieter-Jan Volders ◽  
...  

AbstractThe human transcriptome consists of various RNA biotypes including multiple types of non-coding RNAs (ncRNAs). Current ncRNA compendia remain incomplete partially because they are almost exclusively derived from the interrogation of small- and polyadenylated RNAs. Here, we present a more comprehensive atlas of the human transcriptome that is derived from matching polyA-, total-, and small-RNA profiles of a heterogeneous collection of nearly 300 human tissues and cell lines. We report on thousands of novel RNA species across all major RNA biotypes, including a hitherto poorly-cataloged class of non-polyadenylated single-exon long non-coding RNAs. In addition, we exploit intron abundance estimates from total RNA-sequencing to test and verify functional regulation by novel non-coding RNAs. Our study represents a substantial expansion of the current catalogue of human ncRNAs and their regulatory interactions. All data, analyses, and results are available in the R2 web portal and serve as a basis to further explore RNA biology and function.


2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 20096-20096
Author(s):  
G. Vansant ◽  
K. Oades ◽  
M. Pickering ◽  
J. Monforte

20096 Background: The purpose of this study was to determine the potential of gene signature development to also identify leads among different tumor types compared to normal cells. The theory is that cancer cells will express unique markers that are related to their growth and metastatic potential and therefore be potential targets for therapeutics. Methods: Multiplex PCR Approximately 100 genes were chosen to produce 3 multiplexes for RT-PCR analysis. These genes were identified from gene signatures found in peer reviewed manuscripts. RT and PCR primer pairs designed for each gene to be monitored are chimeric, with a gene specific sequence and a universal sequence common to all forward and reverse primers, respectively. A pair of universal primers that recognize the universal sequences in the chimeric primers are included in the reaction in great excess, with one fluorescently labeled. After reverse transcription and a few rounds of PCR, these universal primers drive the reactions, so all the PCR products are essentially amplified by the universal primer set. The chimeric primers are designed to produce PCR products that all have a difference in length of approximately 5 base pairs, resulting in a stratified set of labeled PCR products. 25ng of total RNA from each tumor derived cell line was used as template for the reverse transcription reactions. Half of these reactions were carried over as templates for the PCR reactions. The PCR products were fluorescently labeled during the PCR reaction and analyzed on a capillary electrophoresis system. Results: Many genes were expressed at high levels across all cancer cell types compared to control cells including an A kinase anchoring protein, cyclin-E2, JPO1, mucin 1, and the transferrin receptor CD 71. Conclusions: Analysis of the expression of genes that are part of diagnostic and prognostic gene signatures in tumor related cell lines revealed many genes whose expression were altered in tumor derived cells that are potentially related to the ability of the carcinogenic cells to proliferate and metastasize. Subsequently, a secondary benefit of the development of prognostic or diagnostic gene signatures with statistical analysis, may be the elucidation of potential therapeutic targets for the treatment of cancer. No significant financial relationships to disclose.


1985 ◽  
Vol 54 (02) ◽  
pp. 533-538 ◽  
Author(s):  
Wilfried Thiel ◽  
Ulrich Delvos ◽  
Gert Müller-Berghaus

SummaryA quantitative determination of soluble fibrin in plasma was carried out by affinity chromatography. For this purpose, desAA-fibrin and fibrinogen immobilized on Sepharose 4B were used at the stationary side whereas batroxobin-induced 125I-desAA-fibrin or thrombin-induced 125I-desAABB-fibrin mixed with plasma containing 131I-fibrinogen represented the fluid phase. The binding characteristics of these mixtures to the immobilized proteins were compared at 20° C and 37° C. Complete binding of both types of fibrin to the immobilized desAA-fibrin was always seen at 20° C as well as at 37° C. However, binding of soluble fibrin was accompanied by substantial binding of fibrinogen that was more pronounced at 20° C. Striking differences depending on the temperature at which the affinity chromatography was carried out, were documented for the fibrinogen-fibrin interaction. At 20° C more than 90% of the applied desAA-fibrin was bound to the immobilized fibrinogen whereas at 37° C only a mean of 17% were retained at the fibrinogen-Sepharose column. An opposite finding with regard to the tested temperature was made with the desAABB-fibrin. Nearly complete binding to insolubilized fibrinogen was found at 37° C (95%) but only 58% of the desAABB-fibrin were bound at 20° C. The binding patterns did not change when the experiments were performed in the presence of calcium ions. The opposite behaviour of the two types of soluble fibrin to immobilized fibrinogen at the different temperatures, together with the substantial binding of fibrinogen in the presence of soluble fibrin to insolubilized fibrin in every setting tested, devaluates affinity chromatography as a tool in the quantitative assessment of soluble fibrin in patients’ plasma.


Viruses ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 1017
Author(s):  
Hirohisa Mekata ◽  
Tomohiro Okagawa ◽  
Satoru Konnai ◽  
Takayuki Miyazawa

Bovine foamy virus (BFV) is a member of the foamy virus family in cattle. Information on the epidemiology, transmission routes, and whole-genome sequences of BFV is still limited. To understand the characteristics of BFV, this study included a molecular survey in Japan and the determination of the whole-genome sequences of 30 BFV isolates. A total of 30 (3.4%, 30/884) cattle were infected with BFV according to PCR analysis. Cattle less than 48 months old were scarcely infected with this virus, and older animals had a significantly higher rate of infection. To reveal the possibility of vertical transmission, we additionally surveyed 77 pairs of dams and 3-month-old calves in a farm already confirmed to have BFV. We confirmed that one of the calves born from a dam with BFV was infected. Phylogenetic analyses revealed that a novel genotype was spread in Japan. In conclusion, the prevalence of BFV in Japan is relatively low and three genotypes, including a novel genotype, are spread in Japan.


2003 ◽  
Vol 69 (11) ◽  
pp. 6541-6549 ◽  
Author(s):  
Gilbert Thierry Lamothe ◽  
Thierry Putallaz ◽  
Han Joosten ◽  
Joey D. Marugg

ABSTRACT A seminested reverse transcription-PCR method coupled to membrane filtration was optimized to investigate the presence of norovirus (NV) RNA sequences in bottled and natural mineral waters. The recovery of viral particles by filtration varied between 28 and 45%, while the limit of detection of the overall method ranged from 6 to 95 viral particles. The assay was broadly reactive, as shown by the successful detection of 27 different viral strains representing 12 common genotypes of NVs. A total of 718 bottled and natural mineral water samples were investigated, including 640 samples of finished, spring, and line products (mostly 1 to 1.5 liters), collected from 36 different water brands of various types and from diverse geographic origins over a 2-year period. In addition, 78 samples of larger volume (10 and 400 to 500 liters) and environmental swabs were investigated. From the 1,436 analyses that were performed for the detection of NVs belonging to genogroups I and II, 34 samples (2.44%) were presumptively positive by seminested RT-PCR. However, confirmation by DNA sequence analysis revealed that all presumptive positive results were either due to nonspecific amplification or to cross-contamination. In conclusion, these results do not provide any evidence for the presence of NV genome sequences in bottled waters.


2013 ◽  
Vol 76 (1) ◽  
pp. 93-98 ◽  
Author(s):  
FRANCISKA M. SCHETS ◽  
HAROLD H. J. L. van den BERG ◽  
ANA MARIA de RODA HUSMAN

The intestinal parasites Cryptosporidium and Giardia are transmitted by water and food and cause human gastroenteritis. Filter-feeding bivalve mollusks, such as oysters and mussels, filter large volumes of water and thus concentrate such pathogens, which makes these bivalves potential vectors of disease. To assess the risk of infection from consumption of contaminated bivalves, parasite numbers and parasite recovery data are required. A modified immunomagnetic separation (IMS) procedure was used to determine Cryptosporidium oocyst and Giardia cyst numbers in individually homogenized oysters (Crassostrea gigas) and mussels (Mytilus edulis). About 12% of the commercial bivalves were positive, with low (oo)cyst numbers per specimen. The recovery efficiency of the IMS procedure was systematically evaluated. Experiments included seeding of homogenized bivalves and whole animals with 100 to 1,000 (oo)cysts. Both seeding procedures yielded highly variable recovery rates. Median Cryptosporidium recoveries were 7.9 to 21% in oysters and 62% in mussels. Median Giardia recoveries were 10 to 25% in oysters and 110% in mussels. Giardia recovery was significantly higher than Cryptosporidium recovery. (Oo)cysts were less efficiently recovered from seeded whole animals than from seeded homogenates, with median Cryptosporidium recoveries of 5.3% in oysters and 45% in mussels and median Giardia recoveries of 4.0% in oysters and 82% in mussels. Both bivalve homogenate seeding and whole animal seeding yielded higher (oo)cyst recovery in mussels than in oysters, likely because of the presence of less shellfish tissue in IMS when analyzing the smaller mussels compared with the larger oysters, resulting in more efficient (oo)cyst extraction. The data generated in this study may be used in the quantitative assessment of the risk of infection with Cryptosporidium or Giardia associated with the consumption of raw bivalve mollusks. This information may be used for making risk management decisions.


2016 ◽  
Vol 16 (1) ◽  
pp. 50 ◽  
Author(s):  
Florence Piron Prunier ◽  
Mathieu Chouteau ◽  
Annabel Whibley ◽  
Mathieu Joron ◽  
Violaine Llaurens

1986 ◽  
Vol 66 (4) ◽  
pp. 1091-1120 ◽  
Author(s):  
Z. Amit ◽  
Z. H. Galina

In this paper we have examined the phenomenon of stress-induced analgesia. We have described the procedures used to measure analgesia and have suggested that the tests can be designed not only to indicate changes in pain threshold but also to allow for the determination of the capacity to execute adaptive behavior. Aside from enabling the analysis of responses, tests that induce reflexive as well as nonreflexive behavior may have the capacity to separate the more complex aspects of pain such as the possible presence of two components of pain, sensory/discriminative and motivational/affective. These components may be of fundamental importance for any attempt to understand the biological significance of SIA. Our examination of the neurotransmitter and neuropeptide systems has revealed that they are affected by the same manipulations that induce SIA. These amines and perhaps peptides play an integral role in learning, motivation, and performance. We conclude that the functional advantage of a reduction of pain during stressful situations is significant because it allows the animal to react in threatening and perhaps critical situations as if there were no pain. Once the pain system is inhibited, other systems modulate and mediate adaptive responses that expedite the survival of the animal.


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