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1
Greenberg, Eliraz, Hodaya Hochberg-Laufer, Shalev Blanga, Noa Kinor und Yaron Shav-Tal. „Cytoplasmic DNA can be detected by RNA fluorescence in situ hybridization“. Nucleic Acids Research 47, Nr. 18 (24.07.2019): e109-e109. http://dx.doi.org/10.1093/nar/gkz645.
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Abstract Fluorescence in situ hybridization (FISH) can be used for the intracellular detection of DNA or RNA molecules. The detection of DNA sequences by DNA FISH requires the denaturation of the DNA double helix to allow the hybridization of the fluorescent probe with DNA in a single stranded form. These hybridization conditions require high temperature and low pH that can damage RNA, and therefore RNA is not typically detectable by DNA FISH. In contrast, RNA FISH does not require a denaturation step since RNA is single stranded, and therefore DNA molecules are not detectable by RNA FISH. Hence, DNA FISH and RNA FISH are mutually exclusive. In this study, we show that plasmid DNA transiently transfected into cells is readily detectable in the cytoplasm by RNA FISH without need for denaturation, shortly after transfection and for several hours. The plasmids, however, are usually not detectable in the nucleus except when the plasmids are efficiently directed into the nucleus, which may imply a more open packaging state for DNA after transfection. This detection of plasmid DNA in the cytoplasm has implications for RNA FISH experiments and opens a window to study conditions when DNA is present in the cytoplasm.
2
Ferguson, Moira M., und Roy G. Danzmann. „RNA/DNA ratios in white muscle as estimates of growth in rainbow trout held at different temperatures“. Canadian Journal of Zoology 68, Nr. 7 (01.07.1990): 1494–98. http://dx.doi.org/10.1139/z90-221.
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The concentrations of RNA, DNA, and protein in white muscle from 240 uniquely tagged rainbow trout (Oncorhynchus mykiss) held at three temperatures (5, 8 (control), and 11 °C) were measured. Both RNA and RNA/DNA ratios were better predictors of recent length- and weight-specific growth rates than they were of absolute fish size. Furthermore, RNA concentrations were better predictors of growth than RNA/DNA ratios. The strength of the regression between either RNA/DNA ratio or RNA and growth rate did not differ consistently among temperatures. Fish reared at warmer temperatures had lower concentrations of RNA for both a given growth rate and a given DNA concentration compared with cold-reared trout. Warm-reared fish also had lower concentrations of DNA and higher protein/DNA ratios than cold-reared trout when fish size was standardized. The concomitant decrease in both RNA and DNA concentrations resulted in marginally lower RNA/DNA ratios in warm-reared fish.
3
Lai, Lan-Tian, Pin Jie Lee und Li-Feng Zhang. „Immunofluorescence protects RNA signals in simultaneous RNA–DNA FISH“. Experimental Cell Research 319, Nr. 3 (Februar 2013): 46–55. http://dx.doi.org/10.1016/j.yexcr.2012.11.009.
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4
Labh, Shyam Narayan. „RNA: DNA Ratio and Growth Performance of Rohu Labeo rohita (Hamilton) Fed Varied Proportion of Protein Diet during Intensive Aquaculture“. International Journal of Life Sciences 9, Nr. 6 (26.09.2015): 113–22. http://dx.doi.org/10.3126/ijls.v9i6.11585.
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An experiment was conducted at Corona of Agriculture (COA)”, Gunjanagar-3, Chitwan, Nepal to complete a project granted by University Grant Commission (UGC), Second Higher Education Project (SHEP) in 2013. Four (average 380 m2) ponds (W, X, Y and Z) with four different diets as D1 (20%), D2 (30%), D3 (40%) and D4 (50%) protein contents were used to conduct the experiment properly to determine the varied proportion of dietary protein on the growth performance of major carp rohu Labeo Rohita (H) in relation with RNA: DNA ratio. After 12th weeks of culture, average length, average weight and specific growth rate of fish were found significantly (P<0.05) higher in the carp with D3 and D4 diet fed fish. Similar results were observed in average total protein, albumin and globulin contents. RNA content increased rapidly with age. The average RNA content increased highest in D2 and D3 diet fed fish while, DNA content were highest in D3 diet fed fish. RNA: DNA ratio was recorded highest in D2 and D3 diet fed fish. RNA: DNA ratio, an indicator of protein synthesis and have been used to accurately estimate the growth rate and feeding condition of fish hence, as the dose of protein increased RNA and DNA contents also increased with age of carp cultured during experiment. Thus, it was clear from this study that the incorporation of protein in diet enhances the growth of fish regardless of species weight groups and the doses, as the average weight of fish was significantly lower in control diet fed fish as compared to the treated one.
5
Yan, Zhangming, Norman Huang, Weixin Wu, Weizhong Chen, Yiqun Jiang, Jingyao Chen, Xuerui Huang et al. „Genome-wide colocalization of RNA–DNA interactions and fusion RNA pairs“. Proceedings of the National Academy of Sciences 116, Nr. 8 (04.02.2019): 3328–37. http://dx.doi.org/10.1073/pnas.1819788116.
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Fusion transcripts are used as biomarkers in companion diagnoses. Although more than 15,000 fusion RNAs have been identified from diverse cancer types, few common features have been reported. Here, we compared 16,410 fusion transcripts detected in cancer (from a published cohort of 9,966 tumor samples of 33 cancer types) with genome-wide RNA–DNA interactions mapped in two normal, noncancerous cell types [using iMARGI, an enhanced version of the mapping of RNA–genome interactions (MARGI) assay]. Among the top 10 most significant RNA–DNA interactions in normal cells, 5 colocalized with the gene pairs that formed fusion RNAs in cancer. Furthermore, throughout the genome, the frequency of a gene pair to exhibit RNA–DNA interactions is positively correlated with the probability of this gene pair to present documented fusion transcripts in cancer. To test whether RNA–DNA interactions in normal cells are predictive of fusion RNAs, we analyzed these in a validation cohort of 96 lung cancer samples using RNA sequencing (RNA-seq). Thirty-seven of 42 fusion transcripts in the validation cohort were found to exhibit RNA–DNA interactions in normal cells. Finally, by combining RNA-seq, single-molecule RNA FISH, and DNA FISH, we detected a cancer sample with EML4-ALK fusion RNA without forming the EML4-ALK fusion gene. Collectively, these data suggest an RNA-poise model, where spatial proximity of RNA and DNA could poise for the creation of fusion transcripts.
6
Ogata, Motoyuki, Gosuke Hayashi, Anri Ichiu und Akimitsu Okamoto. „l-DNA-tagged fluorescence in situ hybridization for highly sensitive imaging of RNAs in single cells“. Organic & Biomolecular Chemistry 18, Nr. 40 (2020): 8084–88. http://dx.doi.org/10.1039/d0ob01635g.
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l-DNA tagged FISH (LT-FISH), including two-step hybridization processes with a l–d chimera oligonucleotide and a fluorescence-labeled PCR product tethering a l-DNA tag, has realized sensitive RNA detection in fixed cells.
7
Chen, Feng, Min Bai, Xiaowen Cao, Yue Zhao, Jing Xue und Yongxi Zhao. „Click-encoded rolling FISH for visualizing single-cell RNA polyadenylation and structures“. Nucleic Acids Research 47, Nr. 22 (04.10.2019): e145-e145. http://dx.doi.org/10.1093/nar/gkz852.
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Abstract Spatially resolved visualization of RNA processing and structures is important for better studying single-cell RNA function and landscape. However, currently available RNA imaging methods are limited to sequence analysis, and not capable of identifying RNA processing events and structures. Here, we developed click-encoded rolling FISH (ClickerFISH) for visualizing RNA polyadenylation and structures in single cells. In ClickerFISH, RNA 3′ polyadenylation tails, single-stranded and duplex regions are chemically labeled with different clickable DNA barcodes. These barcodes then initiate DNA rolling amplification, generating repetitive templates for FISH to image their subcellular distributions. Combined with single-molecule FISH, the proposed strategy can also obtain quantitative information of RNA of interest. Finally, we found that RNA poly(A) tailing and higher-order structures are spatially organized in a cell type-specific style with cell-to-cell heterogeneity. We also explored their spatiotemporal patterns during cell cycle stages, and revealed the highly dynamic organization especially in S phase. This method will help clarify the spatiotemporal architecture of RNA polyadenylation and structures.
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Staeheli, P., Y. X. Yu, R. Grob und O. Haller. „A double-stranded RNA-inducible fish gene homologous to the murine influenza virus resistance gene Mx“. Molecular and Cellular Biology 9, Nr. 7 (Juli 1989): 3117–21. http://dx.doi.org/10.1128/mcb.9.7.3117-3121.1989.
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We cloned and sequenced a 2.35-kilobase EcoRI fragment of genomic DNA from a local freshwater fish (Perca fluviatilis) that strongly hybridized to probes derived from the murine influenza virus resistance gene Mx. The cloned fish DNA contained blocks of sequences related to Mx gene exons 3 to 8, which appeared to represent exons of a bona fide fish gene because they were separated by intron sequences flanked by consensus splice acceptor and donor sites. Injection of double-stranded RNA into the peritoneal cavity of trouts resulted in 5- to 10-fold elevated levels of two liver mRNAs of about 2.0 to 2.5 kilobases in length that hybridized to the cloned genomic DNA. High sequence similarity between this fish gene and the murine Mx gene, identical exon lengths, and similar inducibilities in vivo by double-stranded RNA indicate that we isolated a fragment of a fish Mx gene.
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Ravikiran, K., und R. S. Kulkarni. „Nucleic Acid Content in Male Fresh Water Fish N. notopterus Exposed to Copper Sulphate“. International Letters of Natural Sciences 33 (Januar 2015): 1–8. http://dx.doi.org/10.18052/www.scipress.com/ilns.33.1.
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The nucleic acid content in different tissues such as brain, liver, kidney & testis of both control and copper sulphate (CuSO4) exposed freshwater fish N. notopterus has been studied. The following observation is made in both control and copper sulphate (CuSO4) exposed fish. The testis contain large amount of DNA in comparison to other tissues. The degree of DNA content in control and copper sulphate (CuSO4) exposed fish testis >liver>brain>kidney. The RNA content also exhibited similar to that of DNA, having higher amount in the testis. The degree of RNA content in control and copper sulphate (Cuso4) exposed fish testis >liver>brain>kidney. The nucleic acid content of tissues get reduced under copper sulphate (CuSO4) exposed in the male freshwater fish N. notopterus indicating copper sulphate as a pollutant effect the nucleic acid content in the tissue.
10
Staeheli, P., Y. X. Yu, R. Grob und O. Haller. „A double-stranded RNA-inducible fish gene homologous to the murine influenza virus resistance gene Mx.“ Molecular and Cellular Biology 9, Nr. 7 (Juli 1989): 3117–21. http://dx.doi.org/10.1128/mcb.9.7.3117.
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We cloned and sequenced a 2.35-kilobase EcoRI fragment of genomic DNA from a local freshwater fish (Perca fluviatilis) that strongly hybridized to probes derived from the murine influenza virus resistance gene Mx. The cloned fish DNA contained blocks of sequences related to Mx gene exons 3 to 8, which appeared to represent exons of a bona fide fish gene because they were separated by intron sequences flanked by consensus splice acceptor and donor sites. Injection of double-stranded RNA into the peritoneal cavity of trouts resulted in 5- to 10-fold elevated levels of two liver mRNAs of about 2.0 to 2.5 kilobases in length that hybridized to the cloned genomic DNA. High sequence similarity between this fish gene and the murine Mx gene, identical exon lengths, and similar inducibilities in vivo by double-stranded RNA indicate that we isolated a fragment of a fish Mx gene.
11
Ravikiran, K., und R. S. Kulkarni. „Nucleic Acid Content in Male Fresh Water Fish <i>N.</i> <i>notopterus</i> Exposed to Copper Sulphate“. International Letters of Natural Sciences 33 (27.01.2015): 1–8. http://dx.doi.org/10.56431/p-d78946.
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The nucleic acid content in different tissues such as brain, liver, kidney & testis of both control and copper sulphate (CuSO4) exposed freshwater fish N. notopterus has been studied. The following observation is made in both control and copper sulphate (CuSO4) exposed fish. The testis contain large amount of DNA in comparison to other tissues. The degree of DNA content in control and copper sulphate (CuSO4) exposed fish testis >liver>brain>kidney. The RNA content also exhibited similar to that of DNA, having higher amount in the testis. The degree of RNA content in control and copper sulphate (Cuso4) exposed fish testis >liver>brain>kidney. The nucleic acid content of tissues get reduced under copper sulphate (CuSO4) exposed in the male freshwater fish N. notopterus indicating copper sulphate as a pollutant effect the nucleic acid content in the tissue.
12
Saleh, Rukisah, Sitti Raehanah Muhamad Shaleh, Saleem Mustafa und Sujjat Al-Azad. „RNA/DNA ratio in milkfish (Chanos Chanos) larvae reared at different stocking densities“. Borneo Journal of Marine Science and Aquaculture (BJoMSA) 3, Nr. 1 (31.07.2019): 13–17. http://dx.doi.org/10.51200/bjomsa.v3i1.1697.
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Stocking density can induce stress in fish in an aquaculture system if not handled properly, and the chronic stress may lead to mortality. Several studies have reported that the capability to deal with a range of stocking densities differs among fish species and maturity stage. Hence, fish larvae may have different resilience to stress from the adult fish. Milkfish larvae were reared in hatchery for 50 days using a recirculating culture system at four different stocking densities (8,12,16 and 20 larvae/liter). The growth performance was not significantly different (P>0.05) except at stocking density of 20 larvae/liter. The highest survival rate (88.04%) was recorded in the system with 8 larvae/liter while the lowest (55.44%) in the culture tank where stocking rate was 20 larvae/liter. The stocking density also influenced the RNA / DNA ratio of the milkfish larvae. The RNA/DNA ratio showed a pattern that was identical with that of sigmoid growth where stocking rate of 8, 12, and 16 larvae/liter gained weight until 30 days of rearing. Highest RNA/DNA ratio was recorded at 16 larvae/liter (2.85±0.004), while the lowest was at 20 larvae/liter (2.25±0.217). Food availability might play a limiting factor that leads to the lower RNA/DNA ratio of larvae reared at a high density due to competition.
13
Schmidt, Brigitte F., Jean Chao, Zhengrong Zhu, Robin L. DeBiasio und Gregory Fisher. „Signal Amplification in the Detection of Single-copy DNA and RNA by Enzyme-catalyzed Deposition (CARD) of the Novel Fluorescent Reporter Substrate Cy3.29-Tyramide“. Journal of Histochemistry & Cytochemistry 45, Nr. 3 (März 1997): 365–73. http://dx.doi.org/10.1177/002215549704500304.
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We demonstrate that the CAtalyzed Reporter Deposition method (CARD), utilizing the novel fluorescent reporter Cy3.29-tyramide, is successful in the Fluorescent In Situ Hybridization (FISH) detection of RNA and single-copy DNA. Histone 4 expression is detected in RNA extracts of S-phase, synchronized HeLa cells by dot-blot analysis. Gene expression of histone 4 in HeLa cells is demonstrated by FISH via CARD, utilizing oligonucleotide probes. Fluorescence intensity measurements on CARD-amplified histone 4 RNA detection showed (a) a 25-fold amplification of the signal brightness by biotinylated oligonucleotide probes and (b) a sixfold amplification of the signal brightness by horseradish peroxidase (HRP)-labeled histone 4 probes vs the directly stained control. The sensitivity of the CARD method is demonstrated by the FISH detection of single-copy DNA on human corneal fibroblast and HeLa S3 interphase nuclei. Chromosomal localization of the single copy DNA is demonstrated on HeLa S3 metaphase chromosome spreads. (J Histochem Cytochem 45:365–373, 1997)
14
Walker, Roland. „VIRAL DNA AND CYTOPLASMIC RNA IN LYMPHOCYSTIS CELLS OF FISH*“. Annals of the New York Academy of Sciences 126, Nr. 1 (16.12.2006): 386–95. http://dx.doi.org/10.1111/j.1749-6632.1965.tb14288.x.
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Wang, Shiao Y., Judith L. Lum, Mark G. Carls und Stanley D. Rice. „Relationship between Growth and Total Nucleic Acids in Juvenile Pink Salmon, Oncorhynchus gorbuscha, Fed Crude Oil Contaminated Food“. Canadian Journal of Fisheries and Aquatic Sciences 50, Nr. 5 (01.05.1993): 996–1001. http://dx.doi.org/10.1139/f93-115.
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Total nucleic acids of juvenile pink salmon, Oncorhynchus gorbuscha, fed crude oil contaminated food were analyzed to determine if nucleic acid measurements can be used to evaluate growth of fish collected at oil spill sites. In general, the nucleic acid concentration (micrograms per milligram dry weight) of salmon fry fed food contaminated with either 0.37 or 2.78 mg crude oil/g food was not significantly affected. However, RNA concentration of fry fed food contaminated with 34.83 mg/g was reduced whereas DNA concentration increased. Results over 8 wk indicate decreased protein synthesis and cell content but maintenance of cell integrity in these fish. Growth was inversely related to the level of crude oil contamination in the food. The significant correlations between measured growth and RNA/DNA ratios and RNA contents (micrograms RNA per millimetre fork length) suggest that nucleic acid measurements can be used to compare growth of fish collected from the field.
16
Hövelmann, F., I. Gaspar, J. Chamiolo, M. Kasper, J. Steffen, A. Ephrussi und O. Seitz. „LNA-enhanced DNA FIT-probes for multicolour RNA imaging“. Chemical Science 7, Nr. 1 (2016): 128–35. http://dx.doi.org/10.1039/c5sc03053f.
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A surrogate nucleobase based on 4,4'-cyanine provided up to 195-fold enhancement of red fluorescence upon complementary RNA binding. A mixture of FIT probes allowed the localization of oskar mRNA and other polyadenylated mRNA molecules in developing oocytes from Drosphila melanogaster by wash-free FISH and STED.
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Ferguson, Moira M., Roy G. Danzmann, Fred W. Allendorf und Kathy L. Knudsen. „Metabolic effects of the expression of a phosphoglucomutase locus in the liver of rainbow trout“. Canadian Journal of Zoology 68, Nr. 7 (01.07.1990): 1499–504. http://dx.doi.org/10.1139/z90-222.
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We examined the lengths, weights, condition factors, and hepatosomatic indices of juvenile rainbow trout (Oncorhynchus mykiss) from four full-sib families, each segregating at the temporal regulatory locus Pgm1-t, and the concentrations of RNA, DNA, and protein in their livers and white muscle. In three families, fish with phosphoglucomutase-1 (PGM1) activity in liver (Pgm1-t(b) fish) are significantly longer than their full-sibs lacking activity for liver PGM1 (Pgm1-t(a) fish). Hepatosomatic indices tend to be higher in the Pgm1-t(b) fish than in their Pgm1-t(a) siblings. RNA/DNA ratios in the liver of Pgm1-t(b) fish are significantly higher than those of Pgm1-t(a) fish in two families and marginal in a third. However, no significant differences were detected in a parallel analysis of nucleic acids and protein in white muscle, where PGM1 is expressed in all fish. In a separate experiment, Pgm1-t(b) fish were significantly heavier in all five families, had significantly higher condition factors in two families, and had marginally lower standardized oxygen consumption rates in three families.
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Suthers, IM, JJ Cleary, SC Battaglene und R. Evans. „Relative RNA Content as a Measure of Condition in Larval and Juvenile Fish“. Marine and Freshwater Research 47, Nr. 2 (1996): 301. http://dx.doi.org/10.1071/mf9960301.
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The RNA:DNA ratio for first-feeding larvae (12 days after hatching, 4-6 mm standard length, SL) of Australian bass (Percichthyidae, Macquaria novemaculeata) exposed to four different feeding regimes over 8 days was found to be insensitive to the level of starvation. An alternative condition index based on residuals derived from the regression of RNA on SL showed significant differences over the course of the experiment; these reflected the four feeding treatments. Field collections of juvenile monacanthids (Paramonacanthus otisensis, 10-30 mm SL) from a local estuary revealed no significant difference in RNA: DNA ratio at three sites over six weeks. A residual-based index (RNA on dry weight) showed parallel fluctuations at all sites; they were positively correlated with water temperature. The RNA : DNA ratio depends on the difference in fluorescence between total nucleic acids (TNA, using thiazole orange) and DNA (using Hoechst 33258) to calculate RNA, as there is no RNA-specific fluorescent dye. The numerator is thus dependent on the denominator, and measurement error may be compounded in the ratio, exacerbating potential variability in the index. Ratios may also be variably correlated with age or size and consequently may erroneously indicate condition or growth in larger and faster-growing fish.
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Tripathi, Ghanshyam, und Priyanka Verma. „Starvation-Induced Impairment of Metabolism in a Freshwater Catfish“. Zeitschrift für Naturforschung C 58, Nr. 5-6 (01.06.2003): 446–51. http://dx.doi.org/10.1515/znc-2003-5-626.
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Abstract Starvation induced changes in citrate synthase (CS), glucose-6-phosphate dehydrogenase (G6-PDH), lactate dehydrogenase (LDH), DNA, RNA, RNA/DNA ratio and protein were studied in the freshwater catfish Clarias batrachus. Starvation gradually decreased the activity of CS, G6-PDH and LDH in brain, liver and skeletal muscle of the freshwater catfish. The maximum reduction in these enzyme activities upto 35-45% was observed after 35 days of fasting. This shows substantial decline in aerobic and biosynthetic capacity during starvation period. DNA, RNA, RNA/DNA ratio and protein contents were also reduced from 40-67% which reflects reduction in an overall capacity of the protein synthesis. Starvation - induced macromolecular changes indicate impairment of metabolism in fish.
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Burness, Gary P., Scot C. Leary, Peter W. Hochachka und Christopher D. Moyes. „Allometric scaling of RNA, DNA, and enzyme levels: an intraspecific study“. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 277, Nr. 4 (01.10.1999): R1164—R1170. http://dx.doi.org/10.1152/ajpregu.1999.277.4.r1164.
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The activities of oxidative and glycolytic enzymes show body size-dependent relationships across a wide variety of taxa; however, the mechanistic basis remains unknown. We sampled white epaxial muscle from rainbow trout ( Oncorhynchus mykiss) spanning a 100-fold range in body mass. We measured activities of enzymes from aerobic and anaerobic metabolic pathways, RNA [total RNA and mRNA, pyruvate kinase (PK), citrate synthase (CS), and MyoD mRNA], and total DNA. Total RNA and DNA showed a biphasic relationship with body size, with a break point occurring after fish reached 1 yr of age. In contrast, total RNA/total DNA was constant across the entire size range. Neither CS activity nor CS mRNA levels scaled with body mass. PK activity and PK mRNA levels increased in parallel in yearling fish only ( r 2 = 0.91, P < 0.01). This suggests that although PK expression is transcriptionally regulated in yearlings, the molecular mechanisms regulating expression change with growth and age. This was supported by a positive correlation between MyoD and PK mRNA levels ( r 2 = 0.17, P < 0.05).
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Chérif, Nadia, Mohamed Zouari, Fatma Amdouni, Marwa Mefteh, Ayoub Ksouri, Balkiss Bouhaouala-Zahar und Noureddine Raouafi. „Direct Amperometric Sensing of Fish Nodavirus RNA Using Gold Nanoparticle/DNA-Based Bioconjugates“. Pathogens 10, Nr. 8 (23.07.2021): 932. http://dx.doi.org/10.3390/pathogens10080932.
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We describe the design of a simple and highly sensitive electrochemical bioanalytical method enabling the direct detection of a conserved RNA region within the capsid protein gene of a fish nodavirus, making use of nanostructured disposable electrodes. To achieve this goal, we select a conserved region within the nodavirus RNA2 segment to design a DNA probe that is tethered to the surface of nanostructured disposable screen-printed electrodes. In a proof-of-principle test, a synthetic RNA sequence is detected based on competitive hybridization between two oligonucleotides (biotinylated reporter DNA and target RNA) complimentary to a thiolated DNA capture probe. The method is further validated using extracted RNA samples obtained from healthy carrier Sparus aurata and clinically infected Dicentrarchus labrax fish specimens. In parallel, the sensitivity of the newly described biosensor is compared with a new real-time RT-PCR protocol. The current differences measured in the negative control and in presence of each concentration of target RNA are used to determine the dynamic range of the assay. We obtain a linear response (R2 = 0.995) over a range of RNA concentrations from 0.1 to 25 pM with a detection limit of 20 fM. The results are in good agreement with the results found by the RT-qPCR. This method provides a promising approach toward a more effective diagnosis and risk assessment of viral diseases in aquaculture.
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Xiao, Lu, Renjie Liao und Jia Guo. „Highly Multiplexed Single-Cell In Situ RNA and DNA Analysis by Consecutive Hybridization“. Molecules 25, Nr. 21 (23.10.2020): 4900. http://dx.doi.org/10.3390/molecules25214900.
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The ability to comprehensively profile nucleic acids in individual cells in their natural spatial contexts is essential to advance our understanding of biology and medicine. Here, we report a novel method for spatial transcriptomics and genomics analysis. In this method, every nucleic acid molecule is detected as a fluorescent spot at its natural cellular location throughout the cycles of consecutive fluorescence in situ hybridization (C-FISH). In each C-FISH cycle, fluorescent oligonucleotide probes hybridize to the probes applied in the previous cycle, and also introduce the binding sites for the next cycle probes. With reiterative cycles of hybridization, imaging and photobleaching, the identities of the varied nucleic acids are determined by their unique color sequences. To demonstrate the feasibility of this method, we show that transcripts or genomic loci in single cells can be unambiguously quantified with 2 fluorophores and 16 C-FISH cycles or with 3 fluorophores and 9 C-FISH cycles. Without any error correction, the error rates obtained using the raw data are close to zero. These results indicate that C-FISH potentially enables tens of thousands (216 = 65,536 or 39 = 19,683) of different transcripts or genomic loci to be precisely profiled in individual cells in situ.
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G. Sunitha Devi, Jagadeshwarlu Rapaka and. „EFFECTS OF SUBLETHAL COPPER EXPOSURE ON NUCLEIC ACID LEVELS IN FISH, OREOCHROMIS MOSSAMBICUS (PETERS)“. Journal Of Advanced Zoology 42, Nr. 01 (30.11.2021): 118–27. http://dx.doi.org/10.17762/jaz.v42i01.9.
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ndustries are the major sources of heavy metal pollution and it is released into water and soil. Heavy metals cause several ill effects to aquatic organisms and environment. In the present study, Fishes were exposed to sub lethal concentrations (1/16, 1/12, 1/8 and 1/4th of 96 h LC50 value) i.e. 3mg/L, 4mg/L, 6mg/L and 12mg/L of Copper Sulphate for four different exposure durations of 10, 20, 30 and 40 days. The Nucleic acids (RNA and DNA) in different tissues such as Muscle, Liver, Gills and Kidney of Copper Sulphate (CuSO4) exposed fish, Oreochromis mossambicus has been studied. Decreased tendency was observed in RNA and an insignificant change was observed in DNA levels in all the vital tissues of fish exposed to Copper Sulphate over control. RNA content was gradually decreased with increased exposure period and the decrease was observed to be directly proportional to increased sublethal concentrations. DNA levels decreased higher concentration (12mg/L) and higher duration (40 days).
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Masny, Peter S., On Ying A. Chan, Jessica C. de Greef, Ulla Bengtsson, Melanie Ehrlich, Rabi Tawil, Leslie F. Lock et al. „Analysis of allele-specific RNA transcription in FSHD by RNA-DNA FISH in single myonuclei“. European Journal of Human Genetics 18, Nr. 4 (04.11.2009): 448–56. http://dx.doi.org/10.1038/ejhg.2009.183.
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G. Sunitha Devi, M. Nagaraju and. „ANABOLIC EFFECT OF 17- METHYLTESTOSTERONE HORMONE ON CERTAIN BIOCHEMICAL PARAMETERS OF FISH, TILAPIA MOSSAMBICA“. Journal Of Advanced Zoology 41, Nr. 01 (30.11.2020): 01–14. http://dx.doi.org/10.17762/jaz.v41i01.14.
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One of the major sources of animal protein for human consumption is a fisheries resource. The demand for fishhas increased in recent years due to population growth and the constant search for a healthy diet. The present study was conducted to assess an anabolic impact of an androgenic hormone, 17-Methyltestosterone (MT) on Protein contentand RNA levels in certain tissues i.e. skeletal muscle, liver and gonad of fish, Tilapia mossambica. The hormone was incorporated in the feed and fed to the fish up to four weeks, in the form of three pelleted diets containing 4, 8 and 16 mg MT/ kg diet along with a fourth control group without hormone. The highest increment in tissue Protein content, RNA and DNA levels was observed under 16 mg MT/ kg diet followed by 8 mg MT/ kg diet and 4 mg MT/ kg diet in skeletal muscle, liver and gonad of fish. The more increase in Protein content, RNA and DNA levels was observed after 28 days of the 17-MT oral route of administration. Hence, it is clear that the anabolic hormones such as 17-MT hormone play an important role in enhancing the tissue protein content for nutritional purposes.There was not much change in DNA levels.
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LaPierre, Lorie A., Donald L. Holzschu, Greg A. Wooster, Paul R. Bowser und James W. Casey. „Two Closely Related but Distinct Retroviruses Are Associated with Walleye Discrete Epidermal Hyperplasia“. Journal of Virology 72, Nr. 4 (01.04.1998): 3484–90. http://dx.doi.org/10.1128/jvi.72.4.3484-3490.1998.
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ABSTRACT Walleye discrete epidermal hyperplasia (WEH) is a hyperproliferative skin disease that is prevalent on adult walleye fish throughout North America. We have identified two retroviruses associated with WEH, designated here as walleye epidermal hyperplasia virus type 1 and type 2 (WEHV1 and WEHV2), that are closely related to one another (77% identity) and to walleye dermal sarcoma virus (64% identity) within the polymerase region. WEHV1 and/or WEHV2 viral DNA was readily detected by PCR in hyperplastic tissue samples, but only low levels of viral DNA were detected in uninvolved skin. Southern blot analysis showed one to three copies of integrated WEHV2 viral DNA in lesions but did not detect WEHV2 viral DNA in uninvolved skin from the same fish. Northern blots detected abundant levels of WEHV1 and/or WEHV2 virion RNA transcripts of approximately 13 kb in hyperplastic tissue, but virion RNA was not observed in uninvolved skin and muscle. These results suggest that WEHV1 and WEHV2 are the causative agents of discrete epidermal hyperplasia.
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Mishra, Arvind, und C. P. M. Tripathi. „Impact of 30 days exposure of whole Paper mill effluent (WRPBILE) on nucleic acid profile in the liver and gonad of freshwater teleost Mystus vittatus during annual reproductive cycle“. Environment Conservation Journal 13, Nr. 1&2 (18.06.2012): 51–56. http://dx.doi.org/10.36953/ecj.2012.131209.
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The present study has been undertaken to investigate the biochemical alterations in teleost fish Mystus vittatus after chronic exposure to sublethal concentrations of paper mill effluent for 30 days. a quantitative estimation of DNA and RNA material was made in liver and gonadal tissues throughout the reproductive cycle of the fish. The biochemical variables studied in the control fish showed the maximum values during the spawning phase as compared to other phases of the reproductive cycle of the fish. The changes produced in the nucleic acid content on account of chronic exposure of the fishes for 30 days to 0.4 (40%) and 0.8(80%) of 96 h LC50 of WRBBILE stress were found to be close dependent, being relatively much higher in case of 0.8 WRPBILE when compared to 0.4 WRPBILE. This phenomenon was observed during the three phases of the annual reproductive cycle of the fish. The DNA as well as RNA contents in liver, testis and ovary tissues showed a reduction in case of both the sublethal concentration of effluent in all the three phases of the reproductive cycle. The changes produced by WRPBILE stress were found to statistically very significant in all the phases except in the case of RNA content of testis during the post spawning phase of the fishes exposed to 0.4 WRPBILE. The present study concludes stress induced depletion might be due to degradation of cells, nuclear material and metabolic dysfunction in response to WRPBILE toxicity in the fish.
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McGurk, Michael D., und Wolfgang C. Kusser. „Comparison of Three Methods of Measuring RNA and DNA Concentrations of Individual Pacific Herring, Clupea pallasi, Larvae“. Canadian Journal of Fisheries and Aquatic Sciences 49, Nr. 5 (01.05.1992): 967–74. http://dx.doi.org/10.1139/f92-107.
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Whole-body concentrations of RNA and DNA of individual Pacific herring, Clupea pallasi, larvae were measured with three methods that rely on the fluorescence of dyes bound to nucleic acids. Two methods use the dye ethidium bromide and do not purify nucleic acids, and one method uses ethidium bromide plus the DNA-specific dye bisbenzimidazole and purifies nucleic acids. Highly significant differences in RNA concentration and RNA–DNA ratios were found between the three methods. Significant differences in RNA concentration and RNA–DNA ratios were also found between yolk sac larvae and non-yolk sac larvae analyzed by one of the two ethidium bromide methods, with yolk sac larvae having the lowest ratios. These results suggest that compounds other than nucleic acids may interfere in the two ethidium bromide methods. Until a standard method for extraction and measurement of nucleic acids of larval fish is established, we recommend the use of techniques that purify nucleic acids.
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Ramesh, Archana, Samuel Koo, Soo Jin Kang, Abhisek Ghosal, Francys Alarcon, Tibor Gyuris, Segun C. Jung et al. „A Novel NGS-Based Simultaneous Detection of DNA and RNA Biomarkers Using Total Nucleic Acid (TNA) for Acute Lymphocytic Leukemia (ALL)“. Blood 138, Supplement 1 (05.11.2021): 1875. http://dx.doi.org/10.1182/blood-2021-148012.
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Abstract Background: Acute Lymphocytic Leukemia (ALL) is the most common childhood cancer and accounts for about a quarter of adult acute leukemias. Current NCCN recommendations for clinical testing for risk stratification and treatment guidance include karyotyping, FISH testing for translocations, and RT-PCR for gene fusions and sequencing for DNA mutations detection. Most NGS based approaches test DNA mutations and RNA fusions separately, thereby requiring higher input material and multiple workflows adding to the cost and turn-around-time. An NGS based assay for the detection of DNA variants (NeoGenomics Heme NGS assay) in heme malignancies using Total Nucleic Acid (TNA) is already available in our clinical laboratory and complements FISH based fusion detection and karyotyping but an integral assay to detect both DNA and RNA alterations with a simple workflow for ALL is needed. Methods: We used TNA or RNA spiked-in with DNA to simulate TNA samples, extracted from 93 bone marrow and peripheral blood samples from patients and healthy donors, along with commercial fusion reference myeloid samples Seraseq Myeloid Fusion RNA Mix (SeraCare Inc.) controls. DNA/RNA libraries were prepared using a custom amplicon based Multimodal NGS panel (Qiagen Inc.) targeting 297 genes and 213 genes (select exons) for DNA and RNA fusion detection, respectively. The enriched dual indexed amplicon libraries were sequenced on an Illumina NovaSeq 6000. The sequence data was processed with a customized bioinformatic pipeline for DNA variant as well as a novel machine learning algorithm for RNA fusion detection. We analyzed sensitivity, specificity, accuracy, reproducibility, and repeatability for clinical use. The DNA variants were orthogonally confirmed using other NGS assays, and the RNA fusions were confirmed on an RNA-seq Archer assay or RT-Sanger confirmation assays. Results: Here, we developed and validated a single tube comprehensive NGS panel using a custom multimodal chemistry that uses TNA as input for simultaneous dual detection of DNA and RNA abnormalities in ALL patients' samples. We performed the analytical validation of our Heme NGS assay for the RNA panel to detect fusions in ALL, using TNA input for comprehensive DNA and RNA mutation detection. The fusion concordance was 95% for the RNA fusion panel. The assay detected BCR-ABL1 (7/7), ETV6-RUNX1 (1/1), KMT2A fusions (4/5), TCF3-PBX1 (1/1), and PCM1-JAK2(1/1). The specificity was determined at 100% using a set of 42 fusion negative samples. The limit of detection (LOD) was analyzed using serial dilutions to up to 3 log reduction (LR) using a the Seraseq Myeloid Fusion sample. The fusions were detected down to 1 LR. The reproducibility was tested using a positive fusion and Seraseq samples across three runs and was reported at 100%. Next, a small cohort of ALL samples (n=8) was included as part of this study to simultaneously evaluate DNA and RNA mutations. We detected pathogenic DNA variants in genes previously reported in ALL that included NOTCH1, PTEN, FLT3, IKZF1, JAK1, JAK2, KRAS, NF1, PAX5, U2AF1, TP53, and also RNA fusion BCR-ABL1, and the results were confirmed by an orthogonal NGS assay (NexCourse and RNA-Seqv1 for fusions). One sample carrying a BCR-ABL1 fusion (detected by RNA panel) also harbored mutations in IKZF1 in DNA (detected by DNA panel) that is reported as unfavorable prognostic biomarker for Ph-Like ALL demonstrating comprehensive panel could identify multiple variants within the same sample, demonstrating the advantage DNA+RNA testing has over the classical single gene FISH/RT-PCR testing for the efficient risk stratification and treatment in ALL patients. Conclusions: In this study, we demonstrated that the single tube TNA based NeoGenomics NGS assay can simultaneously detect the DNA and RNA biomarkers associated with ALL for improved diagnostic and prognostic recommendations. The single-tube assay for detection of both RNA fusions and DNA variants using the same sample could offer comprehensive and cost-effective solution for clinical laboratory test for ALL patient care. This is a promising approach that might be used as a dual DNA/RNA alterations detection on other hematological neoplasia. Disclosures Ramesh: Neo Genomics Laboratories: Current Employment. Koo: Neo Genomics Laboratories: Current Employment. Kang: Neo Genomics Laboratories: Current Employment. Ghosal: NeoGenomics Laboratories: Current Employment. Alarcon: NeoGenomics Laboratories: Current Employment. Gyuris: Neo Genomics Laboratories: Current Employment. Jung: NeoGenomics Laboratories, Inc.: Current Employment. Magnan: NeoGenomics Laboratories, Inc.: Current Employment. Nam: NeoGenomics Laboratories, Inc.: Current Employment. Thomas: NeoGenomics Laboratories, Inc.: Current Employment. Fabunan: NeoGenomics Laboratories, Inc.: Current Employment. Petersen: Neo Genomics Laboratories: Current Employment. Lopez-Diaz: NeoGenomics Laboratories, Inc.: Current Employment. Bender: NeoGenomics Laboratories, Inc.: Current Employment. Agersborg: NeoGenomics Laboratories, Inc.: Current Employment. Ye: Neo Genomics Laboratories: Current Employment. Funari: NeoGenomics Laboratories, Inc.: Current Employment.
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Liu, Dandan, Philip R. Tedbury, Shuiyun Lan, Andrew D. Huber, Maritza N. Puray-Chavez, Juan Ji, Eleftherios Michailidis et al. „Visualization of Positive and Negative Sense Viral RNA for Probing the Mechanism of Direct-Acting Antivirals against Hepatitis C Virus“. Viruses 11, Nr. 11 (08.11.2019): 1039. http://dx.doi.org/10.3390/v11111039.
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RNA viruses are highly successful pathogens and are the causative agents for many important diseases. To fully understand the replication of these viruses it is necessary to address the roles of both positive-strand RNA ((+)RNA) and negative-strand RNA ((−)RNA), and their interplay with viral and host proteins. Here we used branched DNA (bDNA) fluorescence in situ hybridization (FISH) to stain both the abundant (+)RNA and the far less abundant (−)RNA in both hepatitis C virus (HCV)- and Zika virus-infected cells, and combined these analyses with visualization of viral proteins through confocal imaging. We were able to phenotypically examine HCV-infected cells in the presence of uninfected cells and revealed the effect of direct-acting antivirals on HCV (+)RNA, (−)RNA, and protein, within hours of commencing treatment. Herein, we demonstrate that bDNA FISH is a powerful tool for the study of RNA viruses that can provide insights into drug efficacy and mechanism of action.
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Nuryati, Sri, Alimuddin Alimuddin, Sukenda Sukenda, Retno Damayanti Soejoedono, Ayi Santika, Fachriyan Hasmi Pasaribu und Komar Sumantadinata. „Construction of a DNA Vaccine Using Glycoprotein Gene and Its Expression Towards Increasing Survival Rate of KHV-Infected Common Carp (Cyprinus carpio)“. Jurnal Natur Indonesia 13, Nr. 1 (21.11.2012): 47. http://dx.doi.org/10.31258/jnat.13.1.47-52.
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Deoxyribonucleic acid (DNA) vaccine has recently been developed as an alternative vaccine against virus infection.This study was the first step of DNA vaccine development to protect cyprinids including common carp (Cyprinuscarpio) and fancy koi (Cyprinus carpio) from KHV (koi herpesvirus) infection in Indonesia. One of KHV glycoproteingenes, i.e. glycoprotein (GP) was ligated with Japanese medaka (Oryzias latipes) â-actin promoter to generatepAct/GP as a DNA vaccine. Fourty fish in body weight of 10-15 g/fish were individually injected by pAct/GP intomuscle in different dosage of 2.5 μg, 7.5 μg and 12.5 μg/100 μl phosphate buffer saline. Total RNA was extractedfrom the 12.5 μg of pAct/GP-injected fish muscle at 24, 48 and 67 hours post-injection to analyze GP expression byRT-PCR method. Potential of pAct/GP as DNA vaccine was examined by injecting KHV into the 30-days-vaccinatedfish. Both of possitive and negative control fish group were not vaccinated. Possitive control fish group wereinjected with KHV, but negative control fish group were not. KHV-challenged fish were reared for 1 month, and thedeath fish were calculated daily. Result of RT-PCR analysis showed that GP gene expression were detected at 3 dpost-injection. Expression of GP in the vaccinated fish groups helped to improve their survival rate after challengedby KHV. All of fish without DNA vaccination had dead 17 days after KHV injection. The results demonstrated thatpAct/GP had high potency to be used as a DNA vaccine against KHV infection in cyprinids.
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Cook, Marcia, und William H. Lynch. „A Sensitive Nested Reverse Transcriptase PCR Assay To Detect Viable Cells of the Fish Pathogen Renibacterium salmoninarum in Atlantic Salmon (Salmo salarL.)“. Applied and Environmental Microbiology 65, Nr. 7 (01.07.1999): 3042–47. http://dx.doi.org/10.1128/aem.65.7.3042-3047.1999.
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ABSTRACT A nested reverse transcriptase (RT) PCR assay detected mRNA of the salmonid pathogen Renibacterium salmoninarum in samples of RNA extracts of between 1 and 10 cells. Total RNA was extracted from cultured bacteria, Atlantic salmon (Salmo salar L.) kidney tissue and ovarian fluid seeded with the pathogen, and kidney tissue from both experimentally challenged and commercially raised fish. Following DNase treatment, extracted RNA was amplified by both RT PCR and PCR by using primers specific for the gene encoding the major protein antigen of R. salmoninarum. A 349-bp amplicon was detected by polyacrylamide gel electrophoresis and silver stain. Inactivation of cultured bacteria by rifampin or erythromycin produced a loss of nested RT PCR mRNA detection corresponding to a loss of bacterial cell viability determined from plate counts but no loss of DNA detection by PCR. In subclinically diseased fish, nested RT PCR identified similar levels of infected fish as determined by viable pathogen culture. Higher percentages of fish testing positive were generated by PCR, particularly in samples from fish previously subjected to antibiotic chemotherapy where 93% were PCR positive, but only 7% were nested RT PCR and culture positive. PCR can generate false-positive data from amplification of target DNA from nonviable pathogen cells. Therefore, nested RT PCR may prove useful for monitoring cultured Atlantic salmon for the presence of viable R. salmoninarum within a useful time frame, particularly samples from broodstock where antibiotic chemotherapy is used prior to spawning to reduce vertical pathogen transmission.
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Basu, Reelina, Lan-Tian Lai, Zhenyu Meng, Jun Wu, Fangwei Shao und Li-Feng Zhang. „Using Amino-Labeled Nucleotide Probes for Simultaneous Single Molecule RNA-DNA FISH“. PLoS ONE 9, Nr. 9 (16.09.2014): e107425. http://dx.doi.org/10.1371/journal.pone.0107425.
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Koo, Dal-Hoe, Hainan Zhao und Jiming Jiang. „Chromatin-associated transcripts of tandemly repetitive DNA sequences revealed by RNA-FISH“. Chromosome Research 24, Nr. 4 (02.09.2016): 467–80. http://dx.doi.org/10.1007/s10577-016-9537-5.
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Foster, A. R., D. F. Houlihan, S. J. Hall und L. J. Burren. „The effects of temperature acclimation on protein synthesis rates and nucleic acid content of juvenile cod (Gadus morhua L.)“. Canadian Journal of Zoology 70, Nr. 11 (01.11.1992): 2095–102. http://dx.doi.org/10.1139/z92-282.
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Juvenile cod were acclimated to cold (5 °C) and warm (15 °C) water temperatures and fed sandeel at a similar ration size (3% body weight∙day−1) for at least 40 days. After this acclimation period, there were no significant differences in either weight-specific growth rate or weight-specific tissue protein synthesis rates (ventricle, gill, stomach, and intestine) between the cold- and warm-acclimated fish. However, every cold-acclimated tissue examined had a significantly higher RNA concentration (μg RNA∙g tissue−1) than the respective warm-acclimated tissue. Cold-acclimated ventricle and intestine had significantly reduced RNA activities (i.e., translational efficiency, g protein synthesized∙g RNA−1∙day−1) compared with the warm-acclimated tissues. In contrast, the mean RNA activities of cold-acclimated stomach and gill were not significantly different from those of the same tissues in the warm-acclimated fish. These alterations in RNA activity and RNA concentration with temperature acclimation probably represent a thermal compensatory mechanism for protein synthesis and growth in cod at 5 °C. Positive linear relationships were observed between tissue protein synthesis rates and tissue RNA concentrations (μg RNA∙g tissue−1). RNA/protein ratios (μg RNA∙mg protein−1) gave a positive (but statistically insignificant) trend with protein synthesis rates. In contrast, a negative trend (statistically insignificant) was observed between tissue protein synthesis rates and tissue RNA/DNA ratios (μg RNA∙μg DNA−1). The use of RNA measurements as biochemical correlates of growth rate in juvenile cod is discussed.
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King, Patrick J. S., Alberto Saiani, Elena V. Bichenkova und Aline F. Miller. „A de novo self-assembling peptide hydrogel biosensor with covalently immobilised DNA-recognising motifs“. Chemical Communications 52, Nr. 40 (2016): 6697–700. http://dx.doi.org/10.1039/c6cc01433j.
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The conjugate co-assemblies within the peptide-rich fibres leaving oligonucleotide recognition elements exposed on the external surface of the peptide fibre to ‘fish out’ DNA/RNA sequences, leading to a fluorescence response.
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Battersby, Brendan James, und Christopher D. Moyes. „Influence of acclimation temperature on mitochondrial DNA, RNA, and enzymes in skeletal muscle“. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 275, Nr. 3 (01.09.1998): R905—R912. http://dx.doi.org/10.1152/ajpregu.1998.275.3.r905.
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Skeletal muscle fibers typically undergo modifications in their mitochondrial content, concomitant with alterations in oxidative metabolism that occur during the development of muscle fiber and in response to physiological stimuli. We examined how cold acclimation affects the mitochondrial properties of two fish skeletal muscle fiber types and how the regulators of mitochondrial content differed between tissues. After 2 mo of acclimation to either 4 or 18°C, mitochondrial enzyme activities in both red and white muscle were higher in cold-acclimated fish. No significant differences were detected between acclimation temperatures in the abundance of steady-state mitochondrial mRNA (cytochrome- c oxidase 1, subunit 6 of F0F1-ATPase), rRNA (16S), or DNA copy number. Steady-state mRNA for nuclear-encoded respiratory (adenine nucleotide translocase 1) and glycolytic genes showed high interindividual variability, particularly in the cold-acclimated fish. Although mitochondrial enzymes were 10-fold different between the two muscle types, mitochondrial DNA copy number differed only 4-fold. The relative abundance of mitochondrial mRNA and nuclear mRNA in red and white muscle reflected the differences in copy number of their respective genes. These data suggest that the response to physiological stimuli and determination of tissue-specific mitochondrial properties likely result from the regulation of nuclear-encoded genes.
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Fenner, Beau J., Rekha Thiagarajan, Hui Kheng Chua und Jimmy Kwang. „Betanodavirus B2 Is an RNA Interference Antagonist That Facilitates Intracellular Viral RNA Accumulation“. Journal of Virology 80, Nr. 1 (01.01.2006): 85–94. http://dx.doi.org/10.1128/jvi.80.1.85-94.2006.
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ABSTRACT Betanodaviruses are small positive-sense bipartite RNA viruses that infect a wide variety of fish species and are notorious for causing lethal outbreaks in juvenile fish hatcheries worldwide. The function of a small nonstructural protein, B2, encoded by the subgenomic RNA3 of betanodaviruses, has remained obscure. Greasy grouper nervous necrosis virus, a betanodavirus model, was used to develop a facile DNA-based reverse genetics system that recapitulated the virus infection cycle, and we used this system to show that B2 is a small nonstructural protein that is essential for high level accumulation of viral RNA1 after RNA transfection of fish, mammalian, and avian cells. The defect in RNA1 accumulation in a B2 mutant was partially complemented by supplying B2 RNA in trans. Confocal analysis of the cellular distribution of B2 indicated that B2 is able to enter the nucleus and accumulates there during the late stages of GGNNV infection. Using human HeLa cells as a cellular RNA interference model, we found that B2 could efficiently antagonize RNA interference, which is a property shared by the distantly related alphanodavirus B2 proteins. This function provides appears to provide an explanation, at least in part, for why B2 mutant RNA1 is severely impaired in its intracellular accumulation.
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Cuadrado, Angeles, und Nicolás Jouve. „Fluorescent in situ hybridization and C-banding analyses of highly repetitive DNA sequences in the heterochromatin of rye (Secale montanum Guss.) and wheat incorporating S. montanum chromosome segments“. Genome 38, Nr. 4 (01.08.1995): 795–802. http://dx.doi.org/10.1139/g95-101.
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The molecular characterization of C-banded regions of Secale montanum Guss. by means of in situ hybridization was performed in order to provide new information about their chromosome structure relative to cultivated rye, Secale cereale L. Accurate identification of individual chromosomes was achieved using simultaneous and (or) successive fluorescent in situ hybridization (FISH) and C-banding. FISH identification was performed using total rye DNA, three highly repetitive rye DNA sequences (pSc119.2, pSc74, and pSc34), and the ribosomal RNA probes pTa71 (18S, 5.8S, and 26S rDNA) and pTa794 (5S rDNA). FISH was also used to identify the chromosome segment involved in two spontaneous translocation lines recovered from a 'Chinese Spring' – S. montanum wheat–rye addition line. FISH analysis revealed the exact translocation breakpoints and allowed the identification of the transferred rye segments. The value of this type of analysis is discussed.Key words: Secale cereale, Secale montanum, rye, repetitive DNA, fluorescence in situ hybridization.
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Kravchenko, V. M., Yu P. Rud, L. P. Buchatski, V. I. Mel’nik, K. Yu Mogylchak, S. P. Ladan und V. M. Yashchuk. „Spectroscopic Studies of Mosquito Iridescent Virus, its Capsid Proteins, Lipids, and DNA“. Ukrainian Journal of Physics 57, Nr. 2 (15.02.2012): 183. http://dx.doi.org/10.15407/ujpe57.2.183.
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Mosquito iridescent virus (MIV) is an icosahedric lipid-containing virus which affects mosquitos of Aedes, Culex, Culizeta genera. Apart from mosquitos and other insects, iridoviruses cause the mass death of fish and can cause huge losses for industrial fish breedings. The MIV virion consists of a core of the genetic material (double-stranded viral DNA) surrounded by a capsid (icosahedral protein shell) and further encased in a lipid envelope. The aim of the work was to determine the role of MIV virion constituents (lipids, capsid proteins, and viral DNA) in the formation of spectral properties of the whole MIV virions. Measured are UV-Vis absorption, fluorescence, fluorescence excitation, and phosphorescence spectra of MIV virions, their capsid proteins, lipids, and viral DNA dissolved in various buffers. It is shown that the UV absorption of MIV virions is caused by the absorption of all virion constituents such as capsid proteins, lipids, and viral DNA. The fluorescence of MIV virions at room temperature is mainly due to the fluorescence of capsid proteins. The spectra measured at low temperatures make it possible to identify the type of a nucleic acid (DNA or RNA) inside the virion thanks to the fact that the DNA and RNA phosphorescence spectra are radically different.
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Nagler, JJ, M. Krisfalusi und DG Cyr. „Quantification of rainbow trout (Oncorhynchus mykiss) estrogen receptor-alpha messenger RNA and its expression in the ovary during the reproductive cycle“. Journal of Molecular Endocrinology 25, Nr. 2 (01.10.2000): 243–51. http://dx.doi.org/10.1677/jme.0.0250243.
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This study developed a quantitative reverse transcription-polymerase chain reaction (RT-PCR) method to measure estrogen receptor-alpha (ERalpha) mRNA in the rainbow trout (Oncorhynchus mykiss). Using RT-PCR, and primers based on the known ERalpha DNA sequence in this species, cDNA sequences representing most of the protein coding region were obtained from ovary poly A(+) RNA. Using these DNA sequences as probes in Northern blot hybridizations confirmed that a single transcript of 4.2 kilobases in poly A(+) RNA could be detected in liver and ovary RNA. For the quantitative RT-PCR assay an internal standard RNA molecule was produced to control for inherent inter-tube differences in amplification efficiency and permit accurate quantification of ERalpha mRNAs. The quantitative RT-PCR assay proved to be highly specific for ERalpha mRNA with a detection limit of 6.9 fg, which corresponds to 273 fg ERalpha mRNA/microg total RNA. The quantitative RT-PCR assay was used to measure the levels of ERalpha mRNA in ovaries of rainbow trout at different stages of reproductive development. Ovarian ERalpha mRNA expression was found during two distinct periods of reproductive development, in pre-vitellogenic ovaries of fish with ovarian follicle diameters (OFDs) </=100 microm and in mid-vitellogenic ovaries with OFDs >1000 microm. ERalpha mRNA could not be detected in the ovaries of fish with OFDs >100 microm but </=1000 microm. The highest levels of ERalpha mRNA were found in late vitellogenic ovaries of fish with OFDs >2000 microm.
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Chapman, Rebecca J., David R. Ghasemi, Felipe Andreiuolo, Valentina Zschernack, Arnault Tauziede Espariat, Francesca Buttarelli, Felice Giangaspero et al. „EPEN-02. OPTIMISING BIOMARKERS FOR ACCURATE EPENDYMOMA DIAGNOSIS, PROGNOSTICATION AND STRATIFICATION WITHIN INTERNATIONAL CLINICAL TRIALS: A BIOMECA STUDY“. Neuro-Oncology 25, Supplement_1 (01.06.2023): i26—i27. http://dx.doi.org/10.1093/neuonc/noad073.106.
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Abstract Accurate identification of brain tumour molecular subgroups is increasingly important. We aimed to establish the most accurate and reproducible ependymoma subgroup biomarker detection techniques, across 147 cases from International Society of Pediatric Oncology (SIOP) Ependymoma II trial participants, enrolled in the pan-European “Biomarkers of Ependymoma in Children and Adolescents (BIOMECA)” study. Across six European BIOMECA laboratories we evaluated epigenetic profiling (DNA methylation array); immunohistochemistry (IHC) for nuclear p65-RELA, H3K27me3, and Tenascin-C; copy number analysis via FISH and MLPA (1q, CDKN2A), and MIP and DNA methylation array (genome-wide copy number evaluation); analysis of ZFTA- and YAP1-fusions by RT-PCR and sequencing, Nanostring and break-apart FISH. DNA Methylation profiling classified 65.3% (n=96/147) of cases as EPN-PFA and 15% (n=22/147) as ST-ZFTA fusion-positive. Immunohistochemical loss of H3K27me3 was a reproducible and accurate surrogate marker for EPN-PFA (sensitivity 99-100% across three centres). IHC for p65-RELA, FISH, and RNA-based analyses effectively identified ZFTA- and YAP1- fused supratentorial ependymomas. Detection of 1q gain using FISH exhibited only 57% inter-centre concordance and low sensitivity and specificity whilst MIP, MLPA and DNA methylation-based approaches demonstrated greater accuracy. We confirm, in a prospective trial cohort, that H3K27me3 IHC is a robust EPN-PFA biomarker. Tenascin-C should be abandoned as a PFA marker. DNA methylation and MIP arrays are effective tools for copy number analysis of 1q gain, 6q and CDKN2A loss whilst FISH is inadequate. Fusion detection was successful, but rare novel fusions need more extensive technologies. We propose CORE and CORE Plus test sets to guide future diagnostic approaches. CORE tests represent those that can currently be used to stratify and inform clinical trials and diagnosis and include IHC and DNA methylation profiling. CORE Plus tests have additional advantages for challenging cases and for use in the research setting and comprise of MIP and RNA-NGS sequencing.
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Amaral, Valter, Henrique N. Cabral und José Paula. „Implications of habitat-specific growth and physiological condition on juvenile crab population structure“. Marine and Freshwater Research 59, Nr. 8 (2008): 726. http://dx.doi.org/10.1071/mf08006.
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Post-settlement processes can regulate the size and structure of marine invertebrate and fish populations. Faster growth and better physiological condition generally increase the survival potential of early juveniles, being usually associated with structurally complex habitats. Successive cohorts of early juvenile Carcinus maenas were followed in sandy and seagrass (Zostera noltii) habitats in the Mira Estuary, Portugal, to estimate growth and physiological condition (evaluated by RNA/DNA ratio) of juvenile populations. Mean cohort growth was similar in both habitats. However, in the sandy habitat, population size structure progressed to cohorts of larger carapace width (CW) and the RNA/DNA ratio was always higher than in the Z. noltii habitat. In this habitat, cohorts of low CW prevailed throughout and RNA/DNA ratio only increased after ~5.0 mm CW. Higher densities characterising seagrass areas may result in higher competition for resources, limiting growth and condition and leading to dispersal to less populated habitats. Larger juveniles had the best physiological condition, especially early in the season. Seagrass habitats do not necessarily yield enhanced growth rates and physiological condition of early juvenile crabs in relation to sandy areas. Knowledge of such trends is vital to understand distribution and abundance patterns of fish and marine invertebrate populations.
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McLaughlin, Robert L., Moira M. Ferguson und David L. G. Noakes. „Concentrations of nucleic acids and protein as indices of nutritional status for recently emerged brook trout (Salvelinus fontinalis)“. Canadian Journal of Fisheries and Aquatic Sciences 52, Nr. 4 (01.04.1995): 848–54. http://dx.doi.org/10.1139/f95-084.
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We examined whether concentrations of nucleic acids and protein sampled from muscle tissue were useful indicators of nutritional status for recently emerged brook trout (Salvelinus fontinalis). Trout collected from the field were maintained in the laboratory under two food treatments: fed versus food deprived. Concentrations of RNA and DNA, and RNA/DNA ratio did not differ significantly between fed and food-deprived trout, but fed trout had higher concentrations of protein and were 20% heavier at a given fork length than food-deprived trout. Wild trout fed in the laboratory had lower concentrations of RNA, DNA, and protein, and lower RNA/DNA ratios than did trout in the field, but were significantly heavier for their fork length. Tissue concentrations of protein may provide an index of nutritional status for recently emerged brook trout while concentrations of nucleic acids apparently do not. It is important to ensure that concentrations of nucleic acids and protein reflect nutritional status adequately for the species and life stage of interest before using these indices to assess the condition of fish in the field.
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Pfankuche, Vanessa, Kerstin Hahn, Rogier Bodewes, Florian Hansmann, André Habierski, Ann-Kathrin Haverkamp, Stephanie Pfaender et al. „Comparison of Different In Situ Hybridization Techniques for the Detection of Various RNA and DNA Viruses“. Viruses 10, Nr. 7 (20.07.2018): 384. http://dx.doi.org/10.3390/v10070384.
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In situ hybridization (ISH) is a technique to determine potential correlations between viruses and lesions. The aim of the study was to compare ISH techniques for the detection of various viruses in different tissues. Tested RNA viruses include atypical porcine pestivirus (APPV) in the cerebellum of pigs, equine and bovine hepacivirus (EqHV, BovHepV) in the liver of horses and cattle, respectively, and Schmallenberg virus (SBV) in the cerebrum of goats. Examined DNA viruses comprise canine bocavirus 2 (CBoV-2) in the intestine of dogs, porcine bocavirus (PBoV) in the spinal cord of pigs and porcine circovirus 2 (PCV-2) in cerebrum, lymph node, and lung of pigs. ISH with self-designed digoxigenin-labelled RNA probes revealed a positive signal for SBV, CBoV-2, and PCV-2, whereas it was lacking for APPV, BovHepV, EqHV, and PBoV. Commercially produced digoxigenin-labelled DNA probes detected CBoV-2 and PCV-2, but failed to detect PBoV. ISH with a commercially available fluorescent ISH (FISH)-RNA probe mix identified nucleic acids of all tested viruses. The detection rate and the cell-associated positive area using the FISH-RNA probe mix was highest compared to the results using other probes and protocols, representing a major benefit of this method. Nevertheless, there are differences in costs and procedure time.
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Verma, Rita, Atul K. Singh und Kamal Jaiswal. „Preliminary study on diminution level of RNA/DNA ratio in tissue of Labeo rohita by exposure to some endocrine disrupting compounds (EDCs)“. Aceh Journal of Animal Science 1, Nr. 1 (17.04.2016): 16–20. http://dx.doi.org/10.13170/ajas.1.1.3800.
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Effects of EDCs particularly on RNA/DNA ratio are yet to be investigated to manage the effluents in natural waters. We investigated exposure effects of endocrine disrupting compounds (EDCs) phthalic acid ester (PAE) and hexachlorocyclohexane (HCH) on the RNA/DNA ratio in tissue of an Indian major carp Labeo rohita. Fish were exposed to pre-determined sublethal concentrations of phthalic acid ester (Di-methyl phthalate (DMP), di-butyl phthalate (DBP), and di-(2- ethylhexyl) phthalate (DEHP) and also HCH for determining the tissue RNA/DNA ratio after 30, 60 and 90 days of exposure in the doses of 0.2 mg L-1, 0.3 mg L-1, and 0.5 mg L-1 respectively. All these tested chemicals significantly (P0.05) inhibited RNA/DNA ratio. The ratio gradually significantly (P0.05) decreased after DEHP where it was 1.9±0.51 F1, 18=15.8 P=0.014 n=19; in case of DBP it was 1.92±0.62 F1, 20=6.5 P=0.012 n=19 and for HCH it was 0.94±0.21 F1, 18=18.08 P=0.0012 n=19 at treatments concentrations of 0.3 mg L-1 and 0.5 mg L-1, compared to control (2.9±0.2) after 90 days. However, there was no statistical significance (P0.05) in RNA/DNA ratio after the DMP (F1, 20=2.4 P=0.15n=21) treatment.
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Selleslagh, Jonathan, und Rachid Amara. „Effect of starvation on condition and growth of juvenile plaice Pleuronectes platessa: nursery habitat quality assessment during the settlement period“. Journal of the Marine Biological Association of the United Kingdom 93, Nr. 2 (11.05.2012): 479–88. http://dx.doi.org/10.1017/s0025315412000483.
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In order to assess the effect of feeding deprivation on growth and condition of field-caught newly settled juvenile plaice Pleuronectes platessa, a 3 week starvation experiment was conducted and changes in RNA/DNA ratio, Fulton's K condition index and recent otolith growth were measured. Fed ad libitum and starved fish were analysed after 0, 1, 2, 3, 4, 5, 7, 9, 14, 16 and 22 days of starvation. In parallel, field newly settled juvenile plaice were collected weekly over the settlement period (between April and May 2005) in the adjacent intertidal zone of the Canche estuary (eastern English Channel, France) and fish performances were estimated. After starvation day 4, Fulton's K condition index and RNA/DNA ratio of juvenile plaice significantly differed between treatments while recent otolith growth differed between treatments after day 9 of the experiment procedure. Results indicate that the three biological parameters are sensitive indices and can be used to accurately assess nutritional condition of wild juvenile fish. Values of 0.32 for RNA/DNA, 0.83 mg. mm−3 for Fulton's K index, and 3.99 µm for recent otolith daily growth were defined as critical threshold values below which juvenile plaice can be classified as ‘starving'. When comparing these experimental values with those from field-caught newly settled juvenile plaice, less than 1% of wild individuals can be classified as ‘starving' whatever the index. Our results suggest that feeding conditions throughout the settlement period on the Canche intertidal nursery ground are favourable to juvenile plaice development and survival, and hence to the recruitment success.
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Ang, Chieh Hwee, Tertius Tansloan Tuy, Tze Wei Chan, Christopher Tham, Si Jie Khoo, Gee Fung How, Charles Chuah und Hein Than. „Utility of Next-Generation Sequencing in the Detection of RNA Fusion Genes in Myeloid Malignancies in Singapore“. Blood 142, Supplement 1 (28.11.2023): 3666. http://dx.doi.org/10.1182/blood-2023-185775.
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Background Oncogenic fusion genes play an instrumental role in the pathogenesis of haematological malignancies and the identification of these genes is crucial for diagnosis, risk stratification and treatment decisions. Conventional diagnostic methods such as karyotyping and fluorescence in situ hybridization (FISH) are the current standard of care but are limited in the sensitivity and availability of different FISH probes for detection of fusion genes. In our institution, we have routinely integrated a targeted next-generation sequencing (NGS) panel in our diagnostic workflow for patients with suspected myeloid malignancies. Here we aim to review the cases with RNA fusion genes detected via NGS and compare these results with that of conventional diagnostic methods. Methods We analysed bone marrow samples from patients with suspected myeloid malignancies at the time of diagnosis in Singapore General Hospital (SGH). Extracted genomic DNA and RNA were amplified, and library preparation was performed with the Oncomine TM Myeloid Research assay (Thermo Fisher Scientific) to interrogate 40 DNA genes and 29 fusion driver genes. NGS libraries were sequenced on the Ion GeneStudio S5 System (Thermo Fisher Scientific) and analyzed using in-house bioinformatics pipelines and the Ion Reporter software. Limit of detection was determined as 3% allele frequency for DNA variants and 10 -2 for RNA fusions. RNA fusions detected by the NGS panel were consolidated and compared with cytogenetics (karyotyping and FISH) results. Results A total of 436 samples from adult patients with suspected myeloid malignancies were received for NGS testing from October 2021 to March 2023 in SGH, the largest tertiary hospital in Singapore. 306 had paired cytogenetics and NGS testing performed. 27 of the 306 cases (9%) had RNA fusions detected by NGS as summarized in Table 1 and Figure 1; their disease subgroups comprised of 16 acute myeloid leukaemia (AML), 5 chronic myeloid leukaemia (CML), 3 BCR-ABL1-negative myeloproliferative neoplasm (MPN), 2 acute promyelocytic leukaemia (APL) and 1 myelodysplastic syndrome (MDS). Eight AML cases had RUNX1-RUNX1T1.R3R3 fusions, with 6/8 (75%) harbouring additional DNA single nucleotide variants or indel mutations. Two AML cases had CBFB-MYH11 fusions, with 1 associated with KIT D419 deletion, and the other with U2AF1, NF1, and ASXL1 mutations. We also identified 2 adult AML cases (aged 49 and 65 years respectively) with DEK-NUP214.D9N18 fusions, both of which had associated FLT3-ITD mutations. There were 5 cases with KMT2A fusions, with a variety of partners including MLLT3.K9M6 (n=2), ELL.K9E2 (n=1), MLLT1.K8M4 (n=1), and MLLT6.K8M9 (n=1). Two patients with APL had PML-RARA detected for different variants: P3R3 and P6R3. Amongst the 8 MPN cases, 5 had BCR-ABL1 fusions consistent with diagnosis of CML. There was 1 case of MPN-Eo with FLP1L1-PDGFRA fusion, and the remaining 2 with ETV6-ABL1 and KMT2A-ELL.K9E2 rare fusions. The detected RNA fusions had clinical impact in establishing diagnosis (93%), prognosis (96%), and guidance of treatment decisions (89%). Out of the 27 cases with RNA fusions detected by NGS, 23 (85%) had concordant karyotype and FISH results. Four RNA fusions, namely KMT2A-ELL, CBFB-MYH11, FIP1L1-PDGFRA and ETV6-ABL1, were not detected by karyotyping or conventional FISH probes performed in our institution. It likely reflects the sensitivity of the NGS panel in detecting RNA fusions in cases of cryptic translocations. Conclusion We have demonstrated that NGS has a high sensitivity for identification of RNA fusion genes, is complementary to conventional cytogenetics testing, and has vast clinical impact in terms of diagnosis, prognostication and clinical management. We advocate for the integration of NGS with DNA and RNA sequencing into routine investigation of suspected myeloid malignancies for a more precise and comprehensive diagnostic approach.
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Pietsch, Torsten, Gerrit Gielen, Andreas Waha, Evelyn Doerner, Andre O. von Bueren, Christian Vokuhl, Glen Kristiansen und Christof Kramm. „HGG-21. Oncogenic tyrosine kinase gene fusions in infant-type hemispheric gliomas - comparison of RNA- and DNA-based methods for their reliable detection“. Neuro-Oncology 24, Supplement_1 (01.06.2022): i65. http://dx.doi.org/10.1093/neuonc/noac079.236.
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Abstract High-grade diffuse gliomas (HGG) in early childhood are characterized by a more favorable outcome compared to older children. We demonstrated in previous studies that these tumors have stable genomes. Activating tyrosine kinase gene fusions in infant-type hemispheric gliomas represent therapeutic targets. 50 supratentorial HGG occurring in children younger than four years were retrieved from the archives of the Brain Tumor Reference Center, Institute of Neuropathology, Bonn University. DNA and RNA were extracted from FFPE tumor samples. Gene fusions were identified on the DNA level by FISH using break-apart probes for ALK, NTRK1, -2, -3, ROS1 and MET and Molecular Inversion Probe (MIP) methodology. On the RNA level, fusion transcripts were detected by targeted RNA sequencing as well as Nanostring assay with fusion-specific probes. 37 supratentorial HGG occurred in the first year of life, 13 HGG between one and four years. 18 cases showed fusions of ALK to different partners; all occurred in the first year of life (18/37, 48.6%). Fusions of ROS1 were found in 5, MET in 3, NTRK1, -2, -3 in 10 cases. 12 cases showed no and two cases novel fusions. The different methods led to comparable results. Only recurrent fusions with known fusion partners were detectable with fusion sequence-specific Nanostring probes and library construction for targeted RNA sequencing failed in a fraction of cases. Break-apart FISH led to reliable results on the next day, and MIP technology represented the most sensitive method for analysis of FFPE samples. Gene fusions involving the tyrosine kinase genes ALK, MET, ROS1 and NTRK1, -2, -3 occurred in 72% of HGG of young children; most frequent were ALK fusions occurring in tumors of infants. DNA-based MIP technology represented the most robust and sensitive assay. A combination of RNA- and DNA-based methods to detect these fusions with high reliability is recommended.
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Biacchesi, Stéphane, Monique LeBerre, Annie Lamoureux, Yoann Louise, Emilie Lauret, Pierre Boudinot und Michel Brémont. „Mitochondrial Antiviral Signaling Protein Plays a Major Role in Induction of the Fish Innate Immune Response against RNA and DNA Viruses“. Journal of Virology 83, Nr. 16 (27.05.2009): 7815–27. http://dx.doi.org/10.1128/jvi.00404-09.
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ABSTRACT Viral infection triggers host innate immune responses through cellular sensor molecules which activate multiple signaling cascades that induce the production of interferons (IFN) and other cytokines. The recent identification of mammalian cytoplasmic viral RNA sensors, such as retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) and their mitochondrial adaptor, the mitochondrial antiviral signaling protein (MAVS), also called IPS-1, VISA, and Cardif, highlights the significance of these molecules in the induction of IFN. Teleost fish also possess a strong IFN system, but nothing is known concerning the RLRs and their downstream adaptor. In this study, we cloned MAVS cDNAs from several fish species (including salmon and zebrafish) and showed that they were orthologs of mammalian MAVS. We demonstrated that overexpression of these mitochondrial proteins in fish cells led to a constitutive induction of IFN and IFN-stimulated genes (ISGs). MAVS-overexpressing cells were almost fully protected against RNA virus infection, with a strong inhibition of both DNA and RNA virus replication (1,000- and 10,000-fold decreases, respectively). Analyses of MAVS deletion mutants showed that both the N-terminal CARD-like and C-terminal transmembrane domains, but not the central proline-rich region, were indispensable for MAVS signaling function. In addition, we cloned the cDNAs encoding a RIG-I-like molecule from salmonid and cyprinid cell lines. Like the case with MAVS, overexpression of RIG-I CARDs in fish cells led to a strong induction of both IFN and ISGs, conferring on fish cells full protection against RNA virus infection. This report provides the first demonstration that teleost fish possess a functional RLR pathway in which MAVS may play a central role in the induction of the innate immune response.