The Experts below are selected from a list of 1938 Experts worldwide ranked by ideXlab platform
K Ophelkeller - One of the best experts on this subject based on the ideXlab platform.
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quantification of gaeumannomyces graminis var tritici in infected roots and soil using Slot Blot Hybridization
Fungal Biology, 1996Co-Authors: Harvey P Herdina, K OphelkellerAbstract:A Slot-Blot Hybridization assay for quantifying Gaeumannomyces graminis var. tritici , in diseased roots and in soil, using a specific and sensitive DNA probe (pG158), has been developed. The pG158 probe hybridized strongly to pathogenic isolates of G. graminis var. tritici , moderately to G. graminis var. avenae and did not hybridize to non-pathogenic G. graminis var. graminis and to a wide range of other soil fungi. The pG158 contains a dispersed highly repeated sequence which provides the high level of sensitivity needed for a quantitative diagnostic test. The amount of Gaeumannomyces graminis var. tritici in soil can be estimated directly from the test soil without growing the susceptible plants or culturing the fungus. pG158 detects approximately 1·0 ng and 0·3 ng of G. graminis var. tritici DNA in 1 μg total DNA extracted from diseased roots and from highly infested soil organic matter, respectively. Both the high copy clone, pG158 and a single copy clone, pG217, clearly distinguished the three varieties of G. graminis and can be used for accurate intra-specific classifications of G. graminis isolates. The pG158 probe provides a tool for field researchers to determine levels of pathogenic isolates of G. graminis in soils and to study the ecology of this fungus.
Yukio Shimosato - One of the best experts on this subject based on the ideXlab platform.
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The AMeX method: A multipurpose tissue‐processing and paraffin‐embedding method. III. Extraction and purification of RNA and application to Slot‐Blot Hybridization analysis
The Journal of Pathology, 2005Co-Authors: Yuichi Sato, Kiyoshi Mukai, Shuichiroh Furuya, Yukio ShimosatoAbstract:RNA was extracted from tissues processed by a new fixation and paraffin-embedding method (the AMeX method) and examined by Northern Blot analysis and Slot-Blot analysis. The RNA extraction method for AMeX-processed tissue sections after the deparaffinization step was the same as that for fresh materials. The total amount of cellular RNA extracted from AMeX-processed mouse liver tissue was slightly less than that extracted from fresh tissue. In tissues of malignant lymphoma, the total amount of cellular RNA extracted from 25 sections each 20 microns thick was about 1.6-1.8 micrograms/mm2, regardless of the histological subtype and period of storage. The extracted RNA was moderately degraded, and usually could not be used for Northern Blot Hybridization analysis. The intensity of ethidium bromide staining and the Hybridization signals of RNA extracted from AMeX-processed tissues were usually reduced in comparison with RNA from fresh material, but specific signals could be detected by Slot-Blot Hybridization analysis. We have demonstrated previously that the AMeX method preserves high-molecular-weight DNA and various antigens. Since the present study showed that information on mRNA can be obtained from AMeX-processed tissue, the versatility and usefulness of this method were further proven.
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the amex method a multipurpose tissue processing and paraffin embedding method iii extraction and purification of rna and application to Slot Blot Hybridization analysis
The Journal of Pathology, 1991Co-Authors: Yuichi Sato, Kiyoshi Mukai, Shuichiroh Furuya, Yukio ShimosatoAbstract:: RNA was extracted from tissues processed by a new fixation and paraffin-embedding method (the AMeX method) and examined by Northern Blot analysis and Slot-Blot analysis. The RNA extraction method for AMeX-processed tissue sections after the deparaffinization step was the same as that for fresh materials. The total amount of cellular RNA extracted from AMeX-processed mouse liver tissue was slightly less than that extracted from fresh tissue. In tissues of malignant lymphoma, the total amount of cellular RNA extracted from 25 sections each 20 microns thick was about 1.6-1.8 micrograms/mm2, regardless of the histological subtype and period of storage. The extracted RNA was moderately degraded, and usually could not be used for Northern Blot Hybridization analysis. The intensity of ethidium bromide staining and the Hybridization signals of RNA extracted from AMeX-processed tissues were usually reduced in comparison with RNA from fresh material, but specific signals could be detected by Slot-Blot Hybridization analysis. We have demonstrated previously that the AMeX method preserves high-molecular-weight DNA and various antigens. Since the present study showed that information on mRNA can be obtained from AMeX-processed tissue, the versatility and usefulness of this method were further proven.
Harvey P Herdina - One of the best experts on this subject based on the ideXlab platform.
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quantification of gaeumannomyces graminis var tritici in infected roots and soil using Slot Blot Hybridization
Fungal Biology, 1996Co-Authors: Harvey P Herdina, K OphelkellerAbstract:A Slot-Blot Hybridization assay for quantifying Gaeumannomyces graminis var. tritici , in diseased roots and in soil, using a specific and sensitive DNA probe (pG158), has been developed. The pG158 probe hybridized strongly to pathogenic isolates of G. graminis var. tritici , moderately to G. graminis var. avenae and did not hybridize to non-pathogenic G. graminis var. graminis and to a wide range of other soil fungi. The pG158 contains a dispersed highly repeated sequence which provides the high level of sensitivity needed for a quantitative diagnostic test. The amount of Gaeumannomyces graminis var. tritici in soil can be estimated directly from the test soil without growing the susceptible plants or culturing the fungus. pG158 detects approximately 1·0 ng and 0·3 ng of G. graminis var. tritici DNA in 1 μg total DNA extracted from diseased roots and from highly infested soil organic matter, respectively. Both the high copy clone, pG158 and a single copy clone, pG217, clearly distinguished the three varieties of G. graminis and can be used for accurate intra-specific classifications of G. graminis isolates. The pG158 probe provides a tool for field researchers to determine levels of pathogenic isolates of G. graminis in soils and to study the ecology of this fungus.
Yuichi Sato - One of the best experts on this subject based on the ideXlab platform.
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The AMeX method: A multipurpose tissue‐processing and paraffin‐embedding method. III. Extraction and purification of RNA and application to Slot‐Blot Hybridization analysis
The Journal of Pathology, 2005Co-Authors: Yuichi Sato, Kiyoshi Mukai, Shuichiroh Furuya, Yukio ShimosatoAbstract:RNA was extracted from tissues processed by a new fixation and paraffin-embedding method (the AMeX method) and examined by Northern Blot analysis and Slot-Blot analysis. The RNA extraction method for AMeX-processed tissue sections after the deparaffinization step was the same as that for fresh materials. The total amount of cellular RNA extracted from AMeX-processed mouse liver tissue was slightly less than that extracted from fresh tissue. In tissues of malignant lymphoma, the total amount of cellular RNA extracted from 25 sections each 20 microns thick was about 1.6-1.8 micrograms/mm2, regardless of the histological subtype and period of storage. The extracted RNA was moderately degraded, and usually could not be used for Northern Blot Hybridization analysis. The intensity of ethidium bromide staining and the Hybridization signals of RNA extracted from AMeX-processed tissues were usually reduced in comparison with RNA from fresh material, but specific signals could be detected by Slot-Blot Hybridization analysis. We have demonstrated previously that the AMeX method preserves high-molecular-weight DNA and various antigens. Since the present study showed that information on mRNA can be obtained from AMeX-processed tissue, the versatility and usefulness of this method were further proven.
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the amex method a multipurpose tissue processing and paraffin embedding method iii extraction and purification of rna and application to Slot Blot Hybridization analysis
The Journal of Pathology, 1991Co-Authors: Yuichi Sato, Kiyoshi Mukai, Shuichiroh Furuya, Yukio ShimosatoAbstract:: RNA was extracted from tissues processed by a new fixation and paraffin-embedding method (the AMeX method) and examined by Northern Blot analysis and Slot-Blot analysis. The RNA extraction method for AMeX-processed tissue sections after the deparaffinization step was the same as that for fresh materials. The total amount of cellular RNA extracted from AMeX-processed mouse liver tissue was slightly less than that extracted from fresh tissue. In tissues of malignant lymphoma, the total amount of cellular RNA extracted from 25 sections each 20 microns thick was about 1.6-1.8 micrograms/mm2, regardless of the histological subtype and period of storage. The extracted RNA was moderately degraded, and usually could not be used for Northern Blot Hybridization analysis. The intensity of ethidium bromide staining and the Hybridization signals of RNA extracted from AMeX-processed tissues were usually reduced in comparison with RNA from fresh material, but specific signals could be detected by Slot-Blot Hybridization analysis. We have demonstrated previously that the AMeX method preserves high-molecular-weight DNA and various antigens. Since the present study showed that information on mRNA can be obtained from AMeX-processed tissue, the versatility and usefulness of this method were further proven.
Kerstin Sahm - One of the best experts on this subject based on the ideXlab platform.
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Relative abundance of Archaea and Bacteria along a thermal gradient of a shallow-water hydrothermal vent quantified by rRNA Slot-Blot Hybridization.
Microbiology, 2000Co-Authors: Stefan M. Sievert, Wiebke Ziebis, Jan Kuever, Kerstin SahmAbstract:Slot-Blot Hybridization of rRNA with domain-specific oligonucleotide probes targeting the 16S rRNA of Archaea and Bacteria was utilized to assess the relative abundance of these domains along a thermal gradient at a shallow submarine hydrothermal vent near Milos Island (Greece). The highest prokaryotic rRNA concentrations (defined as the sum of bacterial and archaeal rRNA) were found in the uppermost sediment surface (0–20 mm), decreasing strongly with depth. This indicates that the microbial activity was mainly occurring in the surface layer of this hydrothermal vent. Furthermore, rRNA concentrations were higher in regions closer to the vent, suggesting that the hydrothermal activity stimulated microbial activity. Archaea seemed to be a minor component of the microbial community at this vent site, even in the zones with higher temperatures. Bacteria made up at least 78% (mean 95%) of the prokaryotic rRNA. However, along the steepest temperature gradient, the proportion of archaeal rRNA increased. Nevertheless, even in the hottest sediment layer where a quantification was possible (in situ temperature 82 °C) archaeal rRNA made up only 11·9% of the prokaryotic rRNA. This suggests that Archaea were generally of minor importance at this vent site and were probably restricted to a narrow niche. The factors that allow Bacteria to dominate in a high temperature environment that was once believed to be the realm of Archaea remain elusive.
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Relative abundance of Archaea and Bacteria along a thermal gradient of a shallow-water hydrothermal vent quantified by rRNA Slot-Blot Hybridization.
Microbiology (Reading England), 2000Co-Authors: Stefan M. Sievert, Wiebke Ziebis, Jan Kuever, Kerstin SahmAbstract:Slot-Blot Hybridization of rRNA with domain-specific oligonucleotide probes targeting the 16S rRNA of Archaea and Bacteria was utilized to assess the relative abundance of these domains along a thermal gradient at a shallow submarine hydrothermal vent near Milos Island (Greece). The highest prokaryotic rRNA concentrations (defined as the sum of bacterial and archaeal rRNA) were found in the uppermost sediment surface (0-20 mm), decreasing strongly with depth. This indicates that the microbial activity was mainly occurring in the surface layer of this hydrothermal vent. Furthermore, rRNA concentrations were higher in regions closer to the vent, suggesting that the hydrothermal activity stimulated microbial activity. Archaea seemed to be a minor component of the microbial community at this vent site, even in the zones with higher temperatures. Bacteria made up at least 78% (mean 95%) of the prokaryotic rRNA. However, along the steepest temperature gradient, the proportion of archaeal rRNA increased. Nevertheless, even in the hottest sediment layer where a quantification was possible (in situ temperature 82 degrees C) archaeal rRNA made up only 11.9% of the prokaryotic rRNA. This suggests that Archaea were generally of minor importance at this vent site and were probably restricted to a narrow niche. The factors that allow Bacteria to dominate in a high temperature environment that was once believed to be the realm of Archaea remain elusive.
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Sulphate reduction and vertical distribution of sulphate-reducing bacteria quantified by rRNA Slot-Blot Hybridization in a coastal marine sediment.
Environmental Microbiology, 1999Co-Authors: Kerstin Sahm, Barbara J. Macgregor, Bo Barker Jørgensen, David A. StahlAbstract:In the past, enumeration of sulphate-reducing bacteria (SRB) by cultivation-based methods generally contradicted measurements of sulphate reduction, suggesting unrealistically high respiration rates per cell. Here, we report evidence that quantification of SRB rRNA by Slot-Blot Hybridization is a valuable tool for a more realistic assessment of SRB abundance in the natural environment. The distribution of SRB was investigated in a coastal marine sediment by Hybridization of membrane-immobilized rRNA with oligonucleotide probes. As represented by general probe-target groups, SRB rRNA contributed between 18% and 25% to the prokaryotic rRNA pool. The dominant SRB were related to complete oxidizing genera (Desulphococcus, Desulphosarcina and Desulphobacterium), while Desulphobacter could not be detected. The vertical profile and quantity of rRNA from SRB was compared with sulphate reduction rates (SRR) measured with 35SO4(2-) tracer in whole-core incubations. While SRB abundance was highest near the surface, peaking at around 1.5 cm, measured sulphate reduction rates were lowest in this region. A second peak of SRB rRNA was observed at the transition zone from oxidized to reduced sediment, directly above the sulphate reduction maximum. Cell numbers calculated by converting the relative contribution of SRB rRNA to the percentage of DAPI-stained cells indicated a population size for SRB of 2.4-6.1 x 10(8) cells cm(-3) wet sediment. Cellular sulphate reduction rates calculated on the basis of these estimated cell numbers were between 0.01 and 0.09 fmol SO4(2-) cell(-1) day(-1), which is below the rates that have been determined for pure cultures (0.2-50 fmol SO4(2-) cell(-1) day(-1)) growing exponentially at nearoptimal temperature with a surplus of substrates.
