The Experts below are selected from a list of 46512 Experts worldwide ranked by ideXlab platform
Jiro Takahara - One of the best experts on this subject based on the ideXlab platform.
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quantitative and sensitive Northern Blot hybridization using pcr generated dna probes labeled with digoxigenin by nick translation
BioTechniques, 1993Co-Authors: Makoto Sato, Koji Murao, Mutsuhiko Mizobuchi, Jiro TakaharaAbstract:Northern Blot hydridization is one of the most convenient methods of detecting an mRNA. Nonradioactive Northern Blotting using digoxigenin (DIG) is becoming widely applied because it is rapid and safe. Previous studies have indicated that DIG-labeled RNA probes are suitable for Northern Blot hybridization. Here, the application of PCR-generated double-stranded DNA probes labeled with DIG by nick translation is described. DNA probes were synthesized by PCR, then labeled with DIG by nick translation
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Quantitative and sensitive Northern Blot hybridization using PCR-generated DNA probes labeled with digoxigenin by nick translation.
BioTechniques, 1993Co-Authors: Makoto Sato, Koji Murao, Mutsuhiko Mizobuchi, Jiro TakaharaAbstract:Northern Blot hybridization is one of the most convenient methods of detecting an mRNA. Nonradioactive Northern Blotting using digoxigenin (DIG) is becoming widely applied because it is rapid and safe. Previous studies have indicated that DIG-labeled RNA probes are suitable for Northern Blot hybridization. Here, the application of PCR-generated double-stranded DNA probes labeled with DIG by nick translation is described. DNA probes were synthesized by PCR, then labeled with DIG by nick translation. Northern Blot hybridization was performed using the DIG-labeled DNA probes, and the signals were detected by means of a chemiluminescent reaction. A low amount of DIG-dUTP in the labeling reaction resulted in excellent Northern Blots with low background. Densitometric analysis of the Blots showed that the mRNA concentrations could be determined by densitometric analysis. The sensitivity of the DIG-Northern system was comparable to Northern Blotting using 32P and was sufficiently sensitive to detect low-abundance mRNA.
Makoto Sato - One of the best experts on this subject based on the ideXlab platform.
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quantitative and sensitive Northern Blot hybridization using pcr generated dna probes labeled with digoxigenin by nick translation
BioTechniques, 1993Co-Authors: Makoto Sato, Koji Murao, Mutsuhiko Mizobuchi, Jiro TakaharaAbstract:Northern Blot hydridization is one of the most convenient methods of detecting an mRNA. Nonradioactive Northern Blotting using digoxigenin (DIG) is becoming widely applied because it is rapid and safe. Previous studies have indicated that DIG-labeled RNA probes are suitable for Northern Blot hybridization. Here, the application of PCR-generated double-stranded DNA probes labeled with DIG by nick translation is described. DNA probes were synthesized by PCR, then labeled with DIG by nick translation
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Quantitative and sensitive Northern Blot hybridization using PCR-generated DNA probes labeled with digoxigenin by nick translation.
BioTechniques, 1993Co-Authors: Makoto Sato, Koji Murao, Mutsuhiko Mizobuchi, Jiro TakaharaAbstract:Northern Blot hybridization is one of the most convenient methods of detecting an mRNA. Nonradioactive Northern Blotting using digoxigenin (DIG) is becoming widely applied because it is rapid and safe. Previous studies have indicated that DIG-labeled RNA probes are suitable for Northern Blot hybridization. Here, the application of PCR-generated double-stranded DNA probes labeled with DIG by nick translation is described. DNA probes were synthesized by PCR, then labeled with DIG by nick translation. Northern Blot hybridization was performed using the DIG-labeled DNA probes, and the signals were detected by means of a chemiluminescent reaction. A low amount of DIG-dUTP in the labeling reaction resulted in excellent Northern Blots with low background. Densitometric analysis of the Blots showed that the mRNA concentrations could be determined by densitometric analysis. The sensitivity of the DIG-Northern system was comparable to Northern Blotting using 32P and was sufficiently sensitive to detect low-abundance mRNA.
Motoharu Hayashi - One of the best experts on this subject based on the ideXlab platform.
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Developmental changes in the expression of growth-associated protein-43 mRNA in the monkey thalamus : Northern Blot and in situ hybridization studies
Neuroscience, 2005Co-Authors: Yumi Murata, Noriyuki Higo, Takao Oishi, Akiko Yamashita, Keiji Matsuda, Motoharu HayashiAbstract:The expression of growth-associated protein-43 has been related to axonal elongation and synaptic sprouting. Using the Northern Blot analysis, we investigated the developmental changes of growth-associated protein-43 mRNA in the thalamus of macaque monkeys. The amount of growth-associated protein-43 mRNA was high at embryonic day 125, and decreased at postnatal day 1. It increased again at postnatal day 8, reached its peak value at postnatal days 50-70, and then decreased gradually until postnatal year 1. We previously reported that the amount of growth-associated protein-43 mRNA in the cerebral cortex decreased roughly exponentially during perinatal and postnatal periods and that it approached the asymptote by postnatal day 70 [Oishi T, Higo N, Umino Y, Matsuda K, Hayashi M (1998) Development of GAP-43 mRNA in the macaque cerebral cortex. Dev Brain Res 109:87-97]. The present findings may indicate that extensive synaptic growth of thalamic neurons continues even after that of cortical neurons has finished. We then performed in situ hybridization to investigate whether the expression level of growth-associated protein-43 mRNA was different among various thalamic nuclei. In the infant thalamus (postnatal days 70-90), moderate to intense expression of growth-associated protein-43 mRNA was detected in all thalamic nuclei. Quantitative analysis in the infant thalamus indicated that the expression levels were different between the nuclear groups that are defined by the origin of their afferents. The expression in the first order nuclei, which receive their primary afferent fibers from ascending pathways [Guillery RW (1995) Anatomical evidence concerning the role of the thalamus in corticocortical communication: a brief review. J Anat 187 (Pt 3):583-592], was significantly higher than that in the higher order nuclei. While moderate expression was also detected in the adult dorsal thalamus, the expression in the first order nuclei was almost the same as that in the higher order nuclei. Thus, the in situ hybridization experiments indicated that the transient postnatal increase in the amount of growth-associated protein-43 mRNA, which was shown by the Northern Blot analysis, was mainly attributed to enhanced expression in the first order nuclei during the postnatal period. This may be a molecular basis for environmentally induced modification of thalamocortical synapses.
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Northern Blot and in situ hybridization analyses for the development of myristoylated alanine rich c kinase substrate mrna in the monkey cerebral cortex
Neuroscience, 2004Co-Authors: Noriyuki Higo, Takao Oishi, Yumi Murata, Akiko Yamashita, Keiji Matsuda, Motoharu HayashiAbstract:Myristoylated alanine-rich C-kinase substrate (MARCKS) is a major neuron-specific substrate for protein kinase C, and is involved in both neurite outgrowth and synaptic plasticity. Using both Northern Blot and in situ hybridization techniques, we investigated whether the expression of MARCKS mRNA in the monkey cerebral neocortex and hippocampus changed during the developmental period. In each of four neocortical areas examined, i.e. the prefrontal area (area FD of [Illinois Monographs in the Medical Sciences (1947) 1]), the temporal association area (TE), the primary somatosensory area (PB), and the primary visual area (OC), the Northern Blot analysis showed that the amount of MARCKS mRNA was high during the fetal and early postnatal periods, and decreased sharply between postnatal day 70 and postnatal month 6. The in situ hybridization experiments showed that the expression of MARCKS mRNA was decreased in every layer of neocortical areas at postnatal month 6 or later. In the primary sensory areas (areas PB and OC), the degree of decrease was higher in the supragranular layers (layers II and III) than in the infragranular layers (layers V and VI). In the hippocampus, the developmental change in the amount of MARCKS mRNA was small, but the in situ hybridization revealed a prominent decrease in Ammon's horn in monkeys on postnatal month 8 and later. These findings indicate that region-specific expression of MARCKS mRNA is established around postnatal month 6. We suggest that the extensive expression of MARCKS mRNA is one of the molecular bases of high plasticity in the infant cerebral cortex.
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Northern Blot and In Situ Hybridization Analyses of MARCKS mRNA Expression in the Cerebral Cortex of the Macaque Monkey
Cerebral cortex (New York N.Y. : 1991), 2002Co-Authors: Noriyuki Higo, Takao Oishi, Akiko Yamashita, Keiji Matsuda, Motoharu HayashiAbstract:Myristoylated alanine-rich C-kinase substrate (MARCKS) is a major substrate for protein kinase C, and is involved in synaptic plasticity. Using both Northern Blot and in situ hybridization techniques, we investigated whether MARCKS expression varied according to the cerebral region, including the hippocampal formation, or according to the type of neuron. Northern Blot analysis showed that the MARCKS mRNA level was higher in the association areas than in the primary sensory and motor areas of the cerebral neocortex. MARCKS mRNA levels in the hippocampus and the amygdala were as high as those in the association areas. The in situ hybridization experiments confirmed the Northern Blot results and showed the distribution and characteristics of MARCKS mRNA-positive neurons. In the association areas of the neocortex, prominent signals were observed in neurons in layers II-VI. In the primary areas, prominent signals were restricted to neurons in layers IV-VI. In the hippocampus, the most intense hybridization signals were observed in neurons in the granule cell layer of the dentate gyrus. The observed region-specific expression might reflect functional specialization for plasticity in individual regions of the monkey cerebral cortex.
Noriyuki Higo - One of the best experts on this subject based on the ideXlab platform.
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Northern Blot and in situ hybridization analyses for the neurogranin mRNA in the developing monkey cerebral cortex
Brain Research, 2006Co-Authors: Noriyuki Higo, Takao Oishi, Yumi Murata, Akiko Yamashita, Keiji Matsuda, Motohairu HayashiAbstract:Abstract Neurogranin is a postsynaptic substrate for protein kinase C, and its expression is related to dendritic spine development and postsynaptic plasticity. Using both Northern Blot analysis and in situ hybridization techniques, we investigated the developmental changes of neurogranin expression in the monkey cerebral cortex. In each of four neocortical areas examined, i.e., the prefrontal area (area FD of von Bonin and Bailey), the temporal association area (TE), the primary somatosensory area (PB), and the primary visual area (OC), the Northern Blot analysis showed that the amount of neurogranin mRNA was low during the prenatal and perinatal periods until postnatal day 8. It increased during postnatal development and reached its peak value at postnatal day 70 (in area OC) or postnatal month 6 (in area FD, TE, and PB). After that, the amount of neurogranin mRNA in the cerebral neocortex decreased gradually until postnatal years 2–3. The in situ hybridization experiments also showed a transient increase of neurogranin mRNA in the neocortex during postnatal day 70 to postnatal month 6. The transient increase was prominent in layers II and III of areas FD and TE; deep in layer III of area PB; and in layers II, III, and IV of area OC. In the hippocampus, in contrast to the results in the neocortex, the expression of neurogranin mRNA was decreased almost continuously during the postnatal period. The transiently increased expression of neurogranin in the postnatal neocortex may be a molecular basis for the postsynaptic modification of afferent inputs possibly from subcortical structures.
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Developmental changes in the expression of growth-associated protein-43 mRNA in the monkey thalamus : Northern Blot and in situ hybridization studies
Neuroscience, 2005Co-Authors: Yumi Murata, Noriyuki Higo, Takao Oishi, Akiko Yamashita, Keiji Matsuda, Motoharu HayashiAbstract:The expression of growth-associated protein-43 has been related to axonal elongation and synaptic sprouting. Using the Northern Blot analysis, we investigated the developmental changes of growth-associated protein-43 mRNA in the thalamus of macaque monkeys. The amount of growth-associated protein-43 mRNA was high at embryonic day 125, and decreased at postnatal day 1. It increased again at postnatal day 8, reached its peak value at postnatal days 50-70, and then decreased gradually until postnatal year 1. We previously reported that the amount of growth-associated protein-43 mRNA in the cerebral cortex decreased roughly exponentially during perinatal and postnatal periods and that it approached the asymptote by postnatal day 70 [Oishi T, Higo N, Umino Y, Matsuda K, Hayashi M (1998) Development of GAP-43 mRNA in the macaque cerebral cortex. Dev Brain Res 109:87-97]. The present findings may indicate that extensive synaptic growth of thalamic neurons continues even after that of cortical neurons has finished. We then performed in situ hybridization to investigate whether the expression level of growth-associated protein-43 mRNA was different among various thalamic nuclei. In the infant thalamus (postnatal days 70-90), moderate to intense expression of growth-associated protein-43 mRNA was detected in all thalamic nuclei. Quantitative analysis in the infant thalamus indicated that the expression levels were different between the nuclear groups that are defined by the origin of their afferents. The expression in the first order nuclei, which receive their primary afferent fibers from ascending pathways [Guillery RW (1995) Anatomical evidence concerning the role of the thalamus in corticocortical communication: a brief review. J Anat 187 (Pt 3):583-592], was significantly higher than that in the higher order nuclei. While moderate expression was also detected in the adult dorsal thalamus, the expression in the first order nuclei was almost the same as that in the higher order nuclei. Thus, the in situ hybridization experiments indicated that the transient postnatal increase in the amount of growth-associated protein-43 mRNA, which was shown by the Northern Blot analysis, was mainly attributed to enhanced expression in the first order nuclei during the postnatal period. This may be a molecular basis for environmentally induced modification of thalamocortical synapses.
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Northern Blot and in situ hybridization analyses for the development of myristoylated alanine rich c kinase substrate mrna in the monkey cerebral cortex
Neuroscience, 2004Co-Authors: Noriyuki Higo, Takao Oishi, Yumi Murata, Akiko Yamashita, Keiji Matsuda, Motoharu HayashiAbstract:Myristoylated alanine-rich C-kinase substrate (MARCKS) is a major neuron-specific substrate for protein kinase C, and is involved in both neurite outgrowth and synaptic plasticity. Using both Northern Blot and in situ hybridization techniques, we investigated whether the expression of MARCKS mRNA in the monkey cerebral neocortex and hippocampus changed during the developmental period. In each of four neocortical areas examined, i.e. the prefrontal area (area FD of [Illinois Monographs in the Medical Sciences (1947) 1]), the temporal association area (TE), the primary somatosensory area (PB), and the primary visual area (OC), the Northern Blot analysis showed that the amount of MARCKS mRNA was high during the fetal and early postnatal periods, and decreased sharply between postnatal day 70 and postnatal month 6. The in situ hybridization experiments showed that the expression of MARCKS mRNA was decreased in every layer of neocortical areas at postnatal month 6 or later. In the primary sensory areas (areas PB and OC), the degree of decrease was higher in the supragranular layers (layers II and III) than in the infragranular layers (layers V and VI). In the hippocampus, the developmental change in the amount of MARCKS mRNA was small, but the in situ hybridization revealed a prominent decrease in Ammon's horn in monkeys on postnatal month 8 and later. These findings indicate that region-specific expression of MARCKS mRNA is established around postnatal month 6. We suggest that the extensive expression of MARCKS mRNA is one of the molecular bases of high plasticity in the infant cerebral cortex.
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Northern Blot and In Situ Hybridization Analyses of MARCKS mRNA Expression in the Cerebral Cortex of the Macaque Monkey
Cerebral cortex (New York N.Y. : 1991), 2002Co-Authors: Noriyuki Higo, Takao Oishi, Akiko Yamashita, Keiji Matsuda, Motoharu HayashiAbstract:Myristoylated alanine-rich C-kinase substrate (MARCKS) is a major substrate for protein kinase C, and is involved in synaptic plasticity. Using both Northern Blot and in situ hybridization techniques, we investigated whether MARCKS expression varied according to the cerebral region, including the hippocampal formation, or according to the type of neuron. Northern Blot analysis showed that the MARCKS mRNA level was higher in the association areas than in the primary sensory and motor areas of the cerebral neocortex. MARCKS mRNA levels in the hippocampus and the amygdala were as high as those in the association areas. The in situ hybridization experiments confirmed the Northern Blot results and showed the distribution and characteristics of MARCKS mRNA-positive neurons. In the association areas of the neocortex, prominent signals were observed in neurons in layers II-VI. In the primary areas, prominent signals were restricted to neurons in layers IV-VI. In the hippocampus, the most intense hybridization signals were observed in neurons in the granule cell layer of the dentate gyrus. The observed region-specific expression might reflect functional specialization for plasticity in individual regions of the monkey cerebral cortex.
Takao Oishi - One of the best experts on this subject based on the ideXlab platform.
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Northern Blot and in situ hybridization analyses for the neurogranin mRNA in the developing monkey cerebral cortex
Brain Research, 2006Co-Authors: Noriyuki Higo, Takao Oishi, Yumi Murata, Akiko Yamashita, Keiji Matsuda, Motohairu HayashiAbstract:Abstract Neurogranin is a postsynaptic substrate for protein kinase C, and its expression is related to dendritic spine development and postsynaptic plasticity. Using both Northern Blot analysis and in situ hybridization techniques, we investigated the developmental changes of neurogranin expression in the monkey cerebral cortex. In each of four neocortical areas examined, i.e., the prefrontal area (area FD of von Bonin and Bailey), the temporal association area (TE), the primary somatosensory area (PB), and the primary visual area (OC), the Northern Blot analysis showed that the amount of neurogranin mRNA was low during the prenatal and perinatal periods until postnatal day 8. It increased during postnatal development and reached its peak value at postnatal day 70 (in area OC) or postnatal month 6 (in area FD, TE, and PB). After that, the amount of neurogranin mRNA in the cerebral neocortex decreased gradually until postnatal years 2–3. The in situ hybridization experiments also showed a transient increase of neurogranin mRNA in the neocortex during postnatal day 70 to postnatal month 6. The transient increase was prominent in layers II and III of areas FD and TE; deep in layer III of area PB; and in layers II, III, and IV of area OC. In the hippocampus, in contrast to the results in the neocortex, the expression of neurogranin mRNA was decreased almost continuously during the postnatal period. The transiently increased expression of neurogranin in the postnatal neocortex may be a molecular basis for the postsynaptic modification of afferent inputs possibly from subcortical structures.
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Developmental changes in the expression of growth-associated protein-43 mRNA in the monkey thalamus : Northern Blot and in situ hybridization studies
Neuroscience, 2005Co-Authors: Yumi Murata, Noriyuki Higo, Takao Oishi, Akiko Yamashita, Keiji Matsuda, Motoharu HayashiAbstract:The expression of growth-associated protein-43 has been related to axonal elongation and synaptic sprouting. Using the Northern Blot analysis, we investigated the developmental changes of growth-associated protein-43 mRNA in the thalamus of macaque monkeys. The amount of growth-associated protein-43 mRNA was high at embryonic day 125, and decreased at postnatal day 1. It increased again at postnatal day 8, reached its peak value at postnatal days 50-70, and then decreased gradually until postnatal year 1. We previously reported that the amount of growth-associated protein-43 mRNA in the cerebral cortex decreased roughly exponentially during perinatal and postnatal periods and that it approached the asymptote by postnatal day 70 [Oishi T, Higo N, Umino Y, Matsuda K, Hayashi M (1998) Development of GAP-43 mRNA in the macaque cerebral cortex. Dev Brain Res 109:87-97]. The present findings may indicate that extensive synaptic growth of thalamic neurons continues even after that of cortical neurons has finished. We then performed in situ hybridization to investigate whether the expression level of growth-associated protein-43 mRNA was different among various thalamic nuclei. In the infant thalamus (postnatal days 70-90), moderate to intense expression of growth-associated protein-43 mRNA was detected in all thalamic nuclei. Quantitative analysis in the infant thalamus indicated that the expression levels were different between the nuclear groups that are defined by the origin of their afferents. The expression in the first order nuclei, which receive their primary afferent fibers from ascending pathways [Guillery RW (1995) Anatomical evidence concerning the role of the thalamus in corticocortical communication: a brief review. J Anat 187 (Pt 3):583-592], was significantly higher than that in the higher order nuclei. While moderate expression was also detected in the adult dorsal thalamus, the expression in the first order nuclei was almost the same as that in the higher order nuclei. Thus, the in situ hybridization experiments indicated that the transient postnatal increase in the amount of growth-associated protein-43 mRNA, which was shown by the Northern Blot analysis, was mainly attributed to enhanced expression in the first order nuclei during the postnatal period. This may be a molecular basis for environmentally induced modification of thalamocortical synapses.
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Northern Blot and in situ hybridization analyses for the development of myristoylated alanine rich c kinase substrate mrna in the monkey cerebral cortex
Neuroscience, 2004Co-Authors: Noriyuki Higo, Takao Oishi, Yumi Murata, Akiko Yamashita, Keiji Matsuda, Motoharu HayashiAbstract:Myristoylated alanine-rich C-kinase substrate (MARCKS) is a major neuron-specific substrate for protein kinase C, and is involved in both neurite outgrowth and synaptic plasticity. Using both Northern Blot and in situ hybridization techniques, we investigated whether the expression of MARCKS mRNA in the monkey cerebral neocortex and hippocampus changed during the developmental period. In each of four neocortical areas examined, i.e. the prefrontal area (area FD of [Illinois Monographs in the Medical Sciences (1947) 1]), the temporal association area (TE), the primary somatosensory area (PB), and the primary visual area (OC), the Northern Blot analysis showed that the amount of MARCKS mRNA was high during the fetal and early postnatal periods, and decreased sharply between postnatal day 70 and postnatal month 6. The in situ hybridization experiments showed that the expression of MARCKS mRNA was decreased in every layer of neocortical areas at postnatal month 6 or later. In the primary sensory areas (areas PB and OC), the degree of decrease was higher in the supragranular layers (layers II and III) than in the infragranular layers (layers V and VI). In the hippocampus, the developmental change in the amount of MARCKS mRNA was small, but the in situ hybridization revealed a prominent decrease in Ammon's horn in monkeys on postnatal month 8 and later. These findings indicate that region-specific expression of MARCKS mRNA is established around postnatal month 6. We suggest that the extensive expression of MARCKS mRNA is one of the molecular bases of high plasticity in the infant cerebral cortex.
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Northern Blot and In Situ Hybridization Analyses of MARCKS mRNA Expression in the Cerebral Cortex of the Macaque Monkey
Cerebral cortex (New York N.Y. : 1991), 2002Co-Authors: Noriyuki Higo, Takao Oishi, Akiko Yamashita, Keiji Matsuda, Motoharu HayashiAbstract:Myristoylated alanine-rich C-kinase substrate (MARCKS) is a major substrate for protein kinase C, and is involved in synaptic plasticity. Using both Northern Blot and in situ hybridization techniques, we investigated whether MARCKS expression varied according to the cerebral region, including the hippocampal formation, or according to the type of neuron. Northern Blot analysis showed that the MARCKS mRNA level was higher in the association areas than in the primary sensory and motor areas of the cerebral neocortex. MARCKS mRNA levels in the hippocampus and the amygdala were as high as those in the association areas. The in situ hybridization experiments confirmed the Northern Blot results and showed the distribution and characteristics of MARCKS mRNA-positive neurons. In the association areas of the neocortex, prominent signals were observed in neurons in layers II-VI. In the primary areas, prominent signals were restricted to neurons in layers IV-VI. In the hippocampus, the most intense hybridization signals were observed in neurons in the granule cell layer of the dentate gyrus. The observed region-specific expression might reflect functional specialization for plasticity in individual regions of the monkey cerebral cortex.
