Methods for Normalizing and for Identifying Small Nucleic Acids

A method for identifying a species of small nucleic acid in a population of different small nucleic acid species of varying abundance comprising,

• ligating adapters to at least one end of at least some of the small nucleic acids in the population to form a multiplicity of different adapter-modified molecules;
• amplifying at least some of the multiplicity of different adapter-modified molecules with a multiplicity of primers, wherein at least some of the primers comprise a degenerate sequence located at the 3′-end of the primer to generate a normalized population;
• determining the nucleotide sequence of a normalized nucleic acid;
• and
• identifying the corresponding small nucleic acid species.

A method for identifying a miRNA species in a population of different small nucleic acid species of varying abundance comprising,

• ligating a 3′ adapter and a 5′ adapter to at least one end of at least some of the small nucleic acids in the population to form a multiplicity of double different adapter-modified molecules, wherein the adapters comprise a 3′ adapter comprising a first primer-binding site and a restriction enzyme cleavage site, a 5′ adapter comprising a second primer-binding site and a restriction enzyme cleavage site, wherein the 5′ adapter and the 3′ adapter comprise deoxyribonucleotides and ribonucleotides, and wherein at least the terminal nucleotide on the 3′-end of the 5′ adapter comprises a ribonucleotide and at least the terminal nucleotide on the 5′-end of the 3′ adapter comprises a ribonucleotide;
• amplifying at least some of the multiplicity of different double adapter-modified molecules with a formulated relative concentration of primers to generate a normalized population, wherein the formulated relative concentration of primers comprises a multiplicity of primer species each comprising different degenerate sequences at their respective 3′-ends and a corresponding universal primer species, wherein at least one of the degenerate sequences comprises one, two, three, four, five, or six nucleotides, wherein the concentration of the universal primer species is at least ten times greater than the concentration of any one of the primer species comprising a degenerate sequence, and wherein the concentration of the universal primer is greater than the total concentration of the multiplicity of primers comprising different degenerate sequences, and wherein the amplifying comprises a polymerase chain reaction;
• determining the nucleotide sequence of a normalized nucleic acid comprising:
• (a) inserting at least a portion of a normalized nucleic acid into a vector;
• (b) amplifying the vector comprising the insert in a host cell;
• and (c) sequencing at least part of the insert of the amplified vector or its complement;
• and
• identifying the corresponding miRNA species.