Method for enrichment of natural antisense messenger RNA

A method for enrichment of natural antisense messenger RNA (mRNA), comprising:

• generating a population of cDNA from poly A+ mRNA in a sample of RNA using a reverse transcriptase enzyme and a polydeoxythymidine-containing oligonucleotide primer having at its 5′-end a sequence complementary to an amplification primer used in a later step in the method;
• incubating the population of generated cDNA to hybridize a sense cDNA from the population of cDNA with an antisense cDNA from the same population of cDNA, wherein the antisense cDNA has a single-stranded segment complementary to the sense cDNA and hybridizes thereto to form a hybrid molecule with a double-stranded segment;
• treating the hybrid molecule with a DNA polymerase having a 5′ to 3′ polymerase activity and a 3′ to 5′ exonuclease activity to remove any single-stranded non-hybridized segments of the hybrid molecule from 3′ to 5′ and to extend the double-stranded segment of the hybrid molecule 5′ to 3′ over an adjacent single-stranded segment as template, thereby forming a double-stranded molecule;
• amplifying the double-stranded molecule using a thermostable polymerase and an amplification primer complementary to the sequence at the 5′-end of the polydeoxythymidine-containing oligonucleotide primer;
• and
• cloning the amplified double-stranded molecule to enrich for a natural antisense mRNA encoded by the amplified double-stranded molecule.