| US 7,544,471 B2 | ||
| Preparing RNA from a wax-embedded tissue specimen | ||
| Hui Wang, Palo Alto, Calif. (US); and Jeffrey R. Sampson, San Francisco, Calif. (US) | ||
| Assigned to Agilent Technologies, Inc., Santa Clara, Calif. (US) | ||
| Filed on Jul. 30, 2005, as Appl. No. 11/193,271. | ||
| Prior Publication US 2007/0026411 A1, Feb. 01, 2007 | ||
| Int. Cl. C12Q 1/68 (2006.01) | ||
| U.S. Cl. 435—6 | 16 Claims |
| 1. A method of preparing labeled miRNA from a wax-embedded tissue specimen comprising:
removing wax from the wax embedded tissue specimen,
digesting the tissue specimen to provide a digested tissue specimen,
purifying a plurality of miRNAs from the digested tissue specimen to provide a purified miRNA fraction,
treating the purified miRNA fraction with a phosphatase to provide a sample containing a plurality of dephosphorylated miRNAs,
heating the sample to at least 80° C. under conditions including at least 40% DMSO; and
contacting the sample with RNA ligase in the presence of a labeled substrate under conditions sufficient to result in coupling
of the labeled substrate to the miRNA in the sample to provide labeled miRNA, the conditions including a DMSO concentration
in the range from about 20% to about 30%, wherein the labeled substrate comprises an observable label moiety and a nucleotide
moiety, the nucleotide moiety having a 5′ phosphate and a 3′ terminus, the observable label moiety directly attached to the
3′ terminus, wherein the observable label moiety is selected from a chromogenic moiety or a fluorophore.
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