Enzymatic labeling of RNA
First Claim
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1. A method comprising:
- a) heating a sample comprising a plurality of RNAs to at least 70°
C. in a solution containing at least 35% DMSO and then cooling the sample to less than 10°
C.; and
b) contacting said sample with an RNA ligase in the presence of a labeled substrate under conditions sufficient to result in coupling of the labeled substrate to the RNA in the sample to provide labeled RNA, the conditions including a DMSO concentration in the range from 15% to 30%, wherein the labeled substrate comprises a nucleotide moiety having a terminal 3′
-phosphate group and an observable label moiety attached to the nucleotide moiety.
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Abstract
Methods are described in which a sample containing RNA is contacted with an enzyme having an RNA ligation activity in the presence of a labeled substrate to provide labeled RNA. Methods of performing an array analysis of a labeled RNA sample are also described.
64 Citations
20 Claims
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1. A method comprising:
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a) heating a sample comprising a plurality of RNAs to at least 70°
C. in a solution containing at least 35% DMSO and then cooling the sample to less than 10°
C.; andb) contacting said sample with an RNA ligase in the presence of a labeled substrate under conditions sufficient to result in coupling of the labeled substrate to the RNA in the sample to provide labeled RNA, the conditions including a DMSO concentration in the range from 15% to 30%, wherein the labeled substrate comprises a nucleotide moiety having a terminal 3′
-phosphate group and an observable label moiety attached to the nucleotide moiety. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19)
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20. A method comprising:
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a) heating a sample comprising a plurality of RNAs to at least 70°
C. in a solution containing at least 35% DMSO and then cooling the sample to less than 10°
C.; andb) contacting said sample with an RNA ligase in the presence of a labeled substrate under conditions sufficient to result in coupling of the labeled substrate to the RNA in the sample to provide labeled RNA, the conditions including a DMSO concentration in the range from 20% to 30%, wherein the labeled substrate has the structure (I);
Q1-Nuc-Q2-Lnk-Lblwherein; Nuc is a nucleoside moiety having a 5′
-terminal and a 3′
-terminal;Q1 is a 5′
-phosphate group attached to the nucleoside moiety Nuc via the 5′
-terminal of the nucleoside moiety,Q2 is a 3′
-phosphate group attached to the nucleoside moiety Nuc via the 3′
-terminal of the nucleoside moiety;Lnk is a linking group; and Lbl is an observable label moiety.
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Specification