Methods of constructing small RNA libraries and their use for expression profiling of target RNAs

A method for detecting and quantifying an amount of a target RNA in a sample, the method comprising:

• a) hybridizing the target RNA with a target-specific oligonucleotide (TSO) to produce a target-specific oligonucleotide hybridized target RNA;
• wherein the target-specific oligonucleotide is
• (i) shorter than the target RNA, or as long as or shorter than the target RNA wherein the target RNA is a miRNA;
• (ii) substantially complementary to the target RNA sequence;
• and
• (iii) wherein the target-specific oligonucleotide is not immobilized;
• b) ligating a 5′ oligonucleotide adapter to a 5′ end of the target-specific oligonucleotide-hybridized target RNA and/or ligating a 3′ oligonucleotide adapter to a 3′ end of the target-specific oligonucleotide-hybridized target RNA, wherein the ligating comprises performing a splint-independent ligation to produce an adapter-ligated target-specific oligonucleotide-hybridized target RNA, and reverse transcribing the adapter-ligated target-specific oligonucleotide-hybridized target RNA, or a portion thereof, to produce a reverse transcript thereof wherein the target-specific oligonucleotide is immobilized during the ligating, wherein the ligation efficiency of the target RNA is dependent upon the hybridization of the target-specific oligonucleotide to the target RNA;
• c) enzymatically amplifying the reverse transcript, or a portion thereof, to produce an amplified polynucleotide comprising a sequence corresponding to or complementary to the target RNA sequence;
• and
• d) quantitatively detecting an amount of the amplified polynucleotide,
• wherein the amount of the amplified polynucleotide correlates with the amount of the target RNA.