METHODS AND COMPOSITIONS FOR DEPLETING ABUNDANT RNA TRANSCRIPTS
The present invention concerns a system for isolating, depleting, and/or preventing the amplification of a targeted nucleic acid, such as mRNA or rRNA, from a sample comprising targeted and nontargeted nucleic acids.
1 . A method of depleting hemoglobin mRNA in a hemoglobin mRNA-containing sample comprising:
binding a mixture of capture nucleic acids of SEQ ID NO:19-SEQ ID NO:28 to the sample in a reaction mixture; and
removing hemoglobin mRNA bound to the mixture of capture nucleic acids from the reaction mixture.
2 . The method of claim 1 , wherein the binding of the capture nucleic acids to the sample prevents amplification of the hemoglobin mRNA.
3 - 15 . (canceled)
16 . The method of claim 1 , wherein said hemoglobin mRNA is a mammalian hemoglobin mRNA.
17 . The method of claim 16 , wherein said mammalian hemoglobin mRNA is a primate or murine hemoglobin mRNA.
18 - 25 . (canceled)
26 . The method of claim 1 , wherein capture nucleic acids and hemoglobin mRNA are removed from the reaction mixture prior to amplification.
27 . (canceled)
28 . The method of claim 1 , wherein the capture nucleic acids are attached to a solid surface prior to binding to the RNA.
29 . The method of claim 1 , wherein the capture nucleic acids are attached to a solid surface after binding to the RNA.
30 . The method of claim 29 , wherein the capture nucleic acids are attached to the solid surface by covalent binding.
31 . The method of claim 29 , wherein the capture nucleic acids are attached to die solid surface via a biotin/streptavidin system.
32 . The method of claim 29 , wherein the solid surface is a bead, a rod, or a plate.
33 . The method of claim 32 , wherein the solid surface is a bead and the bead comprises a super-paramagnetic material.
34 . (canceled)
35 . The method of claim 33 , further comprising using a magnet to remove the bead from the reaction mixture prior to amplification.
36 - 76 . (canceled)
77 . A kit, in a suitable container, comprising a mixture of capture nucleic acids of SEQ ID NO:19-SEQ ID NO:28 and a super-paramagnetic bead.
78 . The kit of claim 77 wherein said super-paramagnetic bead is coated by streptavidin and each of said capture nucleic acids comprises a biotin moiety.
79 - 84 . (canceled)
85 . The kit of claim 78 , wherein each of said sequences SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28 is bound to a biotin moiety by a triethylene glycol linker.
86 - 90 . (canceled)
91 . A method of preventing poly(dT) primed reverse transcription of a target hemoglobin mRNA in a hemoglobin mRNA-containing sample comprising:
binding to the sample a first primer mix comprising SEQ ID NO:63-SEQ ID NO:73 that is specific to the target hemoglobin mRNA;
binding to the sample a second primer comprising a poly(dT) sequence; and
reverse transcribing the RNA in the hemoglobin mRNA-containing sample to form cDNA;
wherein the first primer mix prevents the reverse transcription of the target hemoglobin mRNA by the second primer.
92 . (canceled)
93 . The method of claim 91 , further comprising extending the first primer mix to form a complementary DNA sequence prior to binding the second primer.
94 . The method of claim 91 , wherein the second primer comprises a RNA polymerase promoter sequence.
95 . The method of claim 94 , wherein the RNA polymerase promoter sequence is a T3 polymerase promoter sequence, a T7 polymerase promoter sequence, or a SP2 polymerase promoter sequence.
96 - 151 . (canceled)
152 . The method of claim 1 wherein the binding is in a reaction mixture comprising tetramethylammonium chloride or tetraethylammonium chloride.
153 . The kit of claim 77 wherein the kit further comprises an isostabilizing agent.
154 . The kit of claim 153 wherein the isostabilizing agent is tetramethylammonium chloride.