Subtractive amplification

A subtractive amplification method for preferentially amplifying target RNA relative to non-target RNA in a sample of tester RNA, the method comprising:

• a) contacting a sample of tester RNA with driver nucleic acids under hybridizing conditions, wherein tester RNA which hybridizes to driver nucleic acids is non-target RNA and is inhibited by the hybridized driver nucleic acids from functioning as a template for reverse transcription, and wherein tester RNA which does not hybridize to driver nucleic acids comprises target RNA;
• b) contacting the target RNA with nucleic acid primers under hybridizing conditions;
• c) reverse transcribing the target RNA using the hybridized primers to form a DNA template complementary to at least a portion of the target RNA;
• d) rendering the DNA template single stranded;
• e) contacting the single stranded DNA template with a promoter template under hybridizing conditions;
• f) forming an extended DNA template comprising an RNA polymerase promoter sequence by extending the 3' end of the DNA template using the hybridized promoter template;
• and
• g) transcribing the extended DNA template of step f) to form synthetic target RNA.

A subtractive amplification method for preferentially amplifying target RNA relative to non-target RNA in a sample of tester RNA, both the target and non-target RNA containing terminal priming sequences for amplification, the method comprising:

• a) contacting a sample of tester RNA with driver nucleic acids under hybridizing conditions, wherein tester RNA which hybridizes to driver nucleic acids is non-target RNA and is inhibited by the hybridized driver nucleic acids from functioning as a template for reverse transcription, and wherein tester RNA which does not hybridize to driver nucleic acids comprises target RNA;
• b) reverse transcribing the target RNA using nucleic acid primers that hybridize to the target RNA to form a DNA template complementary to at least a portion of the target RNA;
• c) forming an extended DNA template comprising a functional double stranded RNA polymerase promoter by rendering the DNA template formed in step b) single stranded and extending the 3' end of the DNA template using a hybridized promoter template;
• and
• d) transcribing the extended DNA template formed in step c) with an RNA polymerase to form one or more copies of synthetic target RNA.