COMPOSITIONS AND METHODS FOR MAKING cDNA LIBRARIES FROM SMALL RNAs

A method of preparing a cDNA library from RNA molecules in a biological sample, comprising the steps of:

• providing a 3′ DNA adapter annealed to a unique DNA oligonucleotide, wherein the unique DNA oligonucleotide comprises a first portion that is complementary to the 3′ DNA adapter and a second portion;
• ligating the 3′ DNA adapter to RNA molecules, wherein the ligating is performed under conditions that optimize the ligation reaction;
• reverse transcribing the RNA molecules in the presence of at least one labeled nucleotide to produce labeled cDNA molecules, wherein the reverse transcribing is performed under conditions that optimize the reverse transcription;
• circularizing the labeled cDNA molecules, wherein the circularizing is performed under conditions that optimize the circularization reaction;
• optionally purifying or isolating the cDNA molecules;
• linearizing and performing a first amplification of the cDNA molecules;
• and
• purifying the amplification product,
• thereby preparing a cDNA library.