Patent ID: 7977050

Claim:
A method of performing multiplexed nucleic acid analysis using a in-vitro transcription amplification and reverse transcription detection reaction of double stranded target nucleic acid molecules in a reaction chamber, comprising: (a) adding the double stranded target nucleic acid molecules and all reagents required to complete in-vitro transcription amplification and reverse transcription detection reaction to the reaction chamber, wherein said reaction chamber is not more than 500 nl in volume and includes encoded microparticles arranged in a substantially planar array, wherein differently-encoded microparticles have different DNA oligonucleotides attached thereto, and said encoded microparticles not being positioned such that particular particle types are in particular locations in the array; and (b) incubating the reaction chamber at a constant temperature for a period of time, whereby, said target double-stranded nucleic acid molecules are converted to antisense RNA via an in-vitro transcription reaction in the reaction chamber and said antisense RNA are captured by said DNA oligonucleotides displayed on said encoded microparticles, and wherein the different oligonucleotides are elongated by more than one nucleotide using labeled dNTPs, rNTPs or ddNTPs via a reverse transcription reaction to generate labeled cDNA, when antisense RNA strands which include RNA subsequences complementary to oligonucleotide subsequences in the different oligonucleotides, are present and anneal to the oligonucleotide subsequences; and wherein the reaction volume is confined through the use of oil or another water immiscible liquid such that the reaction volume is small enough such that amplification of the initial double stranded target by a factor of 10 3 generates sufficient antisense RNA concentration such that it can be detected in step (b) above.