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| author_facet |
ZHAO, XINLIANG YU, YI-TAO ZHAO, XINLIANG YU, YI-TAO |
|---|---|
| author |
ZHAO, XINLIANG YU, YI-TAO |
| spellingShingle |
ZHAO, XINLIANG YU, YI-TAO RNA Detection and quantitation of RNA base modifications Molecular Biology |
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zhao, xinliang |
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ZHAO, XINLIANG YU, YI-TAO 1355-8382 1469-9001 Cold Spring Harbor Laboratory Molecular Biology http://dx.doi.org/10.1261/rna.7110804 <jats:p>Using a new combination of previously published techniques, we developed a method for quantitating modified nucleotides in RNAs. First, an RNA is cleaved with RNase H at the 5′ side of a nucleotide of interest. Next,<jats:sup>32</jats:sup>P is substituted for the phosphate at the 5′ end of this nucleotide. Finally, after nuclease P1 digestion, the released radiolabeled nucleotide is analyzed by thin layer chromatography and quantitated by PhosphorImager. Using this method, we showed that the analysis of a pseudouridine at a specific site within an in vitro synthesized U2 RNA is indeed quantitative. We also applied this technique to cellular U2 RNA isolated from mouse liver, and showed that position U34 is ~90% pseudouridylated. This method, combined with previously described reverse transcription-based methods, constitutes a powerful tool for detecting and quantifying modified nucleotides in RNAs. With minor modifications, this method can serve as an effective assay to study RNA modifying enzymes.</jats:p> Detection and quantitation of RNA base modifications RNA |
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10.1261/rna.7110804 |
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Cold Spring Harbor Laboratory, 2004 |
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Cold Spring Harbor Laboratory, 2004 |
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1355-8382 1469-9001 |
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1355-8382 1469-9001 |
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2004 |
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Cold Spring Harbor Laboratory |
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RNA |
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49 |
| title |
Detection and quantitation of RNA base modifications |
| title_unstemmed |
Detection and quantitation of RNA base modifications |
| title_full |
Detection and quantitation of RNA base modifications |
| title_fullStr |
Detection and quantitation of RNA base modifications |
| title_full_unstemmed |
Detection and quantitation of RNA base modifications |
| title_short |
Detection and quantitation of RNA base modifications |
| title_sort |
detection and quantitation of rna base modifications |
| topic |
Molecular Biology |
| url |
http://dx.doi.org/10.1261/rna.7110804 |
| publishDate |
2004 |
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996-1002 |
| description |
<jats:p>Using a new combination of previously published techniques, we developed a method for quantitating modified nucleotides in RNAs. First, an RNA is cleaved with RNase H at the 5′ side of a nucleotide of interest. Next,<jats:sup>32</jats:sup>P is substituted for the phosphate at the 5′ end of this nucleotide. Finally, after nuclease P1 digestion, the released radiolabeled nucleotide is analyzed by thin layer chromatography and quantitated by PhosphorImager. Using this method, we showed that the analysis of a pseudouridine at a specific site within an in vitro synthesized U2 RNA is indeed quantitative. We also applied this technique to cellular U2 RNA isolated from mouse liver, and showed that position U34 is ~90% pseudouridylated. This method, combined with previously described reverse transcription-based methods, constitutes a powerful tool for detecting and quantifying modified nucleotides in RNAs. With minor modifications, this method can serve as an effective assay to study RNA modifying enzymes.</jats:p> |
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| author | ZHAO, XINLIANG, YU, YI-TAO |
| author_facet | ZHAO, XINLIANG, YU, YI-TAO, ZHAO, XINLIANG, YU, YI-TAO |
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| description | <jats:p>Using a new combination of previously published techniques, we developed a method for quantitating modified nucleotides in RNAs. First, an RNA is cleaved with RNase H at the 5′ side of a nucleotide of interest. Next,<jats:sup>32</jats:sup>P is substituted for the phosphate at the 5′ end of this nucleotide. Finally, after nuclease P1 digestion, the released radiolabeled nucleotide is analyzed by thin layer chromatography and quantitated by PhosphorImager. Using this method, we showed that the analysis of a pseudouridine at a specific site within an in vitro synthesized U2 RNA is indeed quantitative. We also applied this technique to cellular U2 RNA isolated from mouse liver, and showed that position U34 is ~90% pseudouridylated. This method, combined with previously described reverse transcription-based methods, constitutes a powerful tool for detecting and quantifying modified nucleotides in RNAs. With minor modifications, this method can serve as an effective assay to study RNA modifying enzymes.</jats:p> |
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| imprint | Cold Spring Harbor Laboratory, 2004 |
| imprint_str_mv | Cold Spring Harbor Laboratory, 2004 |
| institution | DE-Ch1, DE-L229, DE-D275, DE-Bn3, DE-Brt1, DE-Zwi2, DE-D161, DE-Gla1, DE-Zi4, DE-15, DE-Pl11, DE-Rs1, DE-105, DE-14 |
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| spelling | ZHAO, XINLIANG YU, YI-TAO 1355-8382 1469-9001 Cold Spring Harbor Laboratory Molecular Biology http://dx.doi.org/10.1261/rna.7110804 <jats:p>Using a new combination of previously published techniques, we developed a method for quantitating modified nucleotides in RNAs. First, an RNA is cleaved with RNase H at the 5′ side of a nucleotide of interest. Next,<jats:sup>32</jats:sup>P is substituted for the phosphate at the 5′ end of this nucleotide. Finally, after nuclease P1 digestion, the released radiolabeled nucleotide is analyzed by thin layer chromatography and quantitated by PhosphorImager. Using this method, we showed that the analysis of a pseudouridine at a specific site within an in vitro synthesized U2 RNA is indeed quantitative. We also applied this technique to cellular U2 RNA isolated from mouse liver, and showed that position U34 is ~90% pseudouridylated. This method, combined with previously described reverse transcription-based methods, constitutes a powerful tool for detecting and quantifying modified nucleotides in RNAs. With minor modifications, this method can serve as an effective assay to study RNA modifying enzymes.</jats:p> Detection and quantitation of RNA base modifications RNA |
| spellingShingle | ZHAO, XINLIANG, YU, YI-TAO, RNA, Detection and quantitation of RNA base modifications, Molecular Biology |
| title | Detection and quantitation of RNA base modifications |
| title_full | Detection and quantitation of RNA base modifications |
| title_fullStr | Detection and quantitation of RNA base modifications |
| title_full_unstemmed | Detection and quantitation of RNA base modifications |
| title_short | Detection and quantitation of RNA base modifications |
| title_sort | detection and quantitation of rna base modifications |
| title_unstemmed | Detection and quantitation of RNA base modifications |
| topic | Molecular Biology |
| url | http://dx.doi.org/10.1261/rna.7110804 |