Method of isolating, labeling and profiling small RNAs

Details Method of isolating, labeling and profiling small RNAs Method of isolating, labeling and profiling small RNAs

2005-04-28

2009-06-23

Schnizer, Richard (Department: 1635)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C536S023100, C536S024310

Reexamination Certificate

active

07550583

ABSTRACT:
Disclosed herein is a method of selectively labeling non-messenger RNA molecules by isolating total RNA from a tissue or cell, dissolving the isolated RNA, blocking the 3′ end of the RNA and adding T4 RNA ligase and a labeled nucleic acid adaptor. With the results that the T4 RNA ligase ligates the adaptor only to RNA having a 5′ phosphate group and only small RNA are labeled. A method of labeling the 5′ end of mRNA isolates total RNA from a tissue or cell, dissolving RNA in RNase-free water, removing a 5′ cap structure from the mRNA using tobacco acid pyrophosphatase (TAP), removing the TAP, blocking the 3′ end of the RNA molecules; and ligating an adaptor to the RNA by adding T4 RNA ligase and a labeled DNA or RNA adaptor. In another embodiment, there is disclosed a method of expression profiling small RNA by separating labeled RNA from capped RNA, providing a microarray comprising a plurality of probes hybridizable to small RNA, incubating the labeled small RNA with the microarray, washing unhybridized RNA from the microarray and drying the microarray, staining hybridized RNA on the microarray; and scanning the labeled microarray to determine the identity and quantity of labeling to the various miRNA probe sites and thus providing an expression profile of small RNA.

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