The Experts below are selected from a list of 135096 Experts worldwide ranked by ideXlab platform
Antony M. Jose - One of the best experts on this subject based on the ideXlab platform.
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Removing bias against short sequences enables Northern Blotting to better complement RNA-seq for the study of small RNAs.
Nucleic acids research, 2017Co-Authors: Yun S. Choi, Lanelle O. Edwards, Aubrey Dibello, Antony M. JoseAbstract:Changes in small non-coding RNAs such as micro RNAs (miRNAs) can serve as indicators of disease and can be measured using next-generation sequencing of RNA (RNA-seq). Here, we highlight the need for approaches that complement RNA-seq, discover that Northern Blotting of small RNAs is biased against short sequences and develop a protocol that removes this bias. We found that multiple small RNA-seq datasets from the worm Caenorhabditis elegans had shorter forms of miRNAs that appear to be degradation products that arose during the preparatory steps required for RNA-seq. When using Northern Blotting during these studies, we discovered that miRNA-length probes can have ∼1000-fold bias against detecting even synthetic sequences that are 8 nt shorter. By using shorter probes and by performing hybridization and washes at low temperatures, we greatly reduced this bias to enable nearly equivalent detection of 24 to 14 nt RNAs. Our protocol can discriminate RNAs that differ by a single nucleotide and can detect specific miRNAs present in total RNA from C. elegans and pRNAs in total RNA from bacteria. This improved Northern Blotting is particularly useful to analyze products of RNA processing or turnover, and functional RNAs that are shorter than typical miRNAs.
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Removing bias against short sequences enables Northern Blotting to better complement RNA-seq for the study of small RNAs
2016Co-Authors: Yun S. Choi, Lanelle O. Edwards, Aubrey Dibello, Antony M. JoseAbstract:Changes in small non-coding RNAs such as micro RNAs (miRNAs) can serve as indicators of disease and can be measured using next-generation sequencing of RNA (RNA-seq). Here, we highlight the need for approaches that complement RNA-seq, discover that Northern Blotting of small RNAs is biased against short sequences, and develop a protocol that removes this bias. We found that multiple small RNA-seq datasets from the worm C. elegans had shorter forms of miRNAs that appear to be degradation products that arose during the preparatory steps required for RNA-seq. When using Northern Blotting during these studies, we discovered that miRNA-length probes can have a ~360-fold bias against detecting even synthetic sequences that are 8 nt shorter. By using shorter probes and by performing hybridization and washes at low temperatures, we greatly reduced this bias to enable equivalent detection of 24 nt to 14 nt RNAs. Our protocol can better discriminate RNAs that differ by a single nucleotide and can detect specific miRNAs present in total RNA from C. elegans. This improved Northern Blotting is particularly useful to obtain a measure of small RNA integrity, analyze products of RNA processing or turnover, and analyze functional RNAs that are shorter than typical miRNAs.
Karol Mackey - One of the best experts on this subject based on the ideXlab platform.
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UNIT 4.9 Analysis of RNA by Northern and Slot Blot Hybridization
2004Co-Authors: T Brown, Karol MackeyAbstract:Specific sequences in RNA preparations can be detected by Blotting and hybridization analysis using techniques very similar to those originally developed for DNA. Fractionated RNA is transferred from an agarose gel to a membrane support (Northern Blotting); unfractionated RNA is immobilized by slot or dot Blotting. The resulting blots are studied by hybridization analysis with labeled DNA or RNA probes. Northern Blotting differs from Southern Blotting largely in the initial gel fractionation step. Because they are single-stranded, most RNAs are able to form secondary structures by intramolecular base pairing and must therefore be electrophoresed under denaturing conditions if good separations are to be obtained. Denaturation is achieved either by adding formaldehyde to the gel and loading buffers or by treating the RNA with glyoxal and dimethyl sulfoxide (DMSO) prior to loading. The Basic Protocol describes Blotting and hybridization of RNA fractionated in an agarose-formaldehyde gel. Alternate protocols describe the glyoxal/DMSO method for denaturing gel electrophoresis and slot-blot hybridization of RNA samples. Stripping hybridization probes from blots can be done under three different sets of conditions; these methods are outlined in a Support Protocol.
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Analysis of RNA by Northern and slot blot hybridization.
Current protocols in molecular biology, 2004Co-Authors: T Brown, Karol MackeyAbstract:Specific sequences in RNA preparations can be detected by Blotting and hybridization analysis using techniques very similar to those originally developed for DNA. Fractionated RNA is transferred from an agarose gel to a membrane support (Northern Blotting); unfractionated RNA is immobilized by slot or dot Blotting. The resulting blots are studied by hybridization analysis with labeled DNA or RNA probes. Northern Blotting differs from Southern Blotting largely in the initial gel fractionation step. Because they are single-stranded, most RNAs are able to form secondary structures by intramolecular base pairing and must therefore be electrophoresed under denaturing conditions if good separations are to be obtained. Denaturation is achieved either by adding formaldehyde to the gel and loading buffers or by treating the RNA with glyoxal and dimethyl sulfoxide (DMSO) prior to loading. The Basic Protocol describes Blotting and hybridization of RNA fractionated in an agarose-formaldehyde gel. Alternate Protocols describe the glyoxal/DMSO method for denaturing gel electrophoresis and slot-blot hybridization of RNA samples. Stripping hybridization probes from blots can be done under three different sets of conditions; these methods are outlined in a Support Protocol.
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Current Protocols in Molecular Biology - Analysis of RNA by Northern and Slot‐Blot Hybridization
Current protocols in molecular biology, 2001Co-Authors: T Brown, Karol MackeyAbstract:Specific sequences in RNA preparations can be detected by Blotting and hybridization analysis using techniques very similar to those originally developed for DNA. Fractionated RNA is transferred from an agarose gel to a membrane support (Northern Blotting); unfractionated RNA is immobilized by slot or dot Blotting. The resulting blots are studied by hybridization analysis with labeled DNA or RNA probes. Northern Blotting differs from Southern Blotting largely in the initial gel fractionation step. Because they are single-stranded, most RNAs are able to form secondary structures by intramolecular base pairing and must therefore be electrophoresed under denaturing conditions if good separations are to be obtained. Denaturation is achieved either by adding formaldehyde to the gel and loading buffers or by treating the RNA with glyoxal and dimethyl sulfoxide (DMSO) prior to loading. The Basic Protocol describes Blotting and hybridization of RNA fractionated in an agarose-formaldehyde gel. Alternate Protocols describe the glyoxal/DMSO method for denaturing gel electrophoresis and slot-blot hybridization of RNA samples. Stripping hybridization probes from blots can be done under three different sets of conditions; these methods are outlined in a Support Protocol.
Yun S. Choi - One of the best experts on this subject based on the ideXlab platform.
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Removing bias against short sequences enables Northern Blotting to better complement RNA-seq for the study of small RNAs.
Nucleic acids research, 2017Co-Authors: Yun S. Choi, Lanelle O. Edwards, Aubrey Dibello, Antony M. JoseAbstract:Changes in small non-coding RNAs such as micro RNAs (miRNAs) can serve as indicators of disease and can be measured using next-generation sequencing of RNA (RNA-seq). Here, we highlight the need for approaches that complement RNA-seq, discover that Northern Blotting of small RNAs is biased against short sequences and develop a protocol that removes this bias. We found that multiple small RNA-seq datasets from the worm Caenorhabditis elegans had shorter forms of miRNAs that appear to be degradation products that arose during the preparatory steps required for RNA-seq. When using Northern Blotting during these studies, we discovered that miRNA-length probes can have ∼1000-fold bias against detecting even synthetic sequences that are 8 nt shorter. By using shorter probes and by performing hybridization and washes at low temperatures, we greatly reduced this bias to enable nearly equivalent detection of 24 to 14 nt RNAs. Our protocol can discriminate RNAs that differ by a single nucleotide and can detect specific miRNAs present in total RNA from C. elegans and pRNAs in total RNA from bacteria. This improved Northern Blotting is particularly useful to analyze products of RNA processing or turnover, and functional RNAs that are shorter than typical miRNAs.
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Removing bias against short sequences enables Northern Blotting to better complement RNA-seq for the study of small RNAs
2016Co-Authors: Yun S. Choi, Lanelle O. Edwards, Aubrey Dibello, Antony M. JoseAbstract:Changes in small non-coding RNAs such as micro RNAs (miRNAs) can serve as indicators of disease and can be measured using next-generation sequencing of RNA (RNA-seq). Here, we highlight the need for approaches that complement RNA-seq, discover that Northern Blotting of small RNAs is biased against short sequences, and develop a protocol that removes this bias. We found that multiple small RNA-seq datasets from the worm C. elegans had shorter forms of miRNAs that appear to be degradation products that arose during the preparatory steps required for RNA-seq. When using Northern Blotting during these studies, we discovered that miRNA-length probes can have a ~360-fold bias against detecting even synthetic sequences that are 8 nt shorter. By using shorter probes and by performing hybridization and washes at low temperatures, we greatly reduced this bias to enable equivalent detection of 24 nt to 14 nt RNAs. Our protocol can better discriminate RNAs that differ by a single nucleotide and can detect specific miRNAs present in total RNA from C. elegans. This improved Northern Blotting is particularly useful to obtain a measure of small RNA integrity, analyze products of RNA processing or turnover, and analyze functional RNAs that are shorter than typical miRNAs.
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.
T Brown - One of the best experts on this subject based on the ideXlab platform.
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UNIT 4.9 Analysis of RNA by Northern and Slot Blot Hybridization
2004Co-Authors: T Brown, Karol MackeyAbstract:Specific sequences in RNA preparations can be detected by Blotting and hybridization analysis using techniques very similar to those originally developed for DNA. Fractionated RNA is transferred from an agarose gel to a membrane support (Northern Blotting); unfractionated RNA is immobilized by slot or dot Blotting. The resulting blots are studied by hybridization analysis with labeled DNA or RNA probes. Northern Blotting differs from Southern Blotting largely in the initial gel fractionation step. Because they are single-stranded, most RNAs are able to form secondary structures by intramolecular base pairing and must therefore be electrophoresed under denaturing conditions if good separations are to be obtained. Denaturation is achieved either by adding formaldehyde to the gel and loading buffers or by treating the RNA with glyoxal and dimethyl sulfoxide (DMSO) prior to loading. The Basic Protocol describes Blotting and hybridization of RNA fractionated in an agarose-formaldehyde gel. Alternate protocols describe the glyoxal/DMSO method for denaturing gel electrophoresis and slot-blot hybridization of RNA samples. Stripping hybridization probes from blots can be done under three different sets of conditions; these methods are outlined in a Support Protocol.
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Analysis of RNA by Northern and slot blot hybridization.
Current protocols in molecular biology, 2004Co-Authors: T Brown, Karol MackeyAbstract:Specific sequences in RNA preparations can be detected by Blotting and hybridization analysis using techniques very similar to those originally developed for DNA. Fractionated RNA is transferred from an agarose gel to a membrane support (Northern Blotting); unfractionated RNA is immobilized by slot or dot Blotting. The resulting blots are studied by hybridization analysis with labeled DNA or RNA probes. Northern Blotting differs from Southern Blotting largely in the initial gel fractionation step. Because they are single-stranded, most RNAs are able to form secondary structures by intramolecular base pairing and must therefore be electrophoresed under denaturing conditions if good separations are to be obtained. Denaturation is achieved either by adding formaldehyde to the gel and loading buffers or by treating the RNA with glyoxal and dimethyl sulfoxide (DMSO) prior to loading. The Basic Protocol describes Blotting and hybridization of RNA fractionated in an agarose-formaldehyde gel. Alternate Protocols describe the glyoxal/DMSO method for denaturing gel electrophoresis and slot-blot hybridization of RNA samples. Stripping hybridization probes from blots can be done under three different sets of conditions; these methods are outlined in a Support Protocol.
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Current Protocols in Molecular Biology - Analysis of RNA by Northern and Slot‐Blot Hybridization
Current protocols in molecular biology, 2001Co-Authors: T Brown, Karol MackeyAbstract:Specific sequences in RNA preparations can be detected by Blotting and hybridization analysis using techniques very similar to those originally developed for DNA. Fractionated RNA is transferred from an agarose gel to a membrane support (Northern Blotting); unfractionated RNA is immobilized by slot or dot Blotting. The resulting blots are studied by hybridization analysis with labeled DNA or RNA probes. Northern Blotting differs from Southern Blotting largely in the initial gel fractionation step. Because they are single-stranded, most RNAs are able to form secondary structures by intramolecular base pairing and must therefore be electrophoresed under denaturing conditions if good separations are to be obtained. Denaturation is achieved either by adding formaldehyde to the gel and loading buffers or by treating the RNA with glyoxal and dimethyl sulfoxide (DMSO) prior to loading. The Basic Protocol describes Blotting and hybridization of RNA fractionated in an agarose-formaldehyde gel. Alternate Protocols describe the glyoxal/DMSO method for denaturing gel electrophoresis and slot-blot hybridization of RNA samples. Stripping hybridization probes from blots can be done under three different sets of conditions; these methods are outlined in a Support Protocol.
