Patent ID: 7465562

Claim:
A single-container method for synthesizing cDNA from RNA in a biological sample using a reverse transcriptase and amplifying and detecting at least one selected sequence of said cDNA, comprising: a) combining said biological sample with a disruption reagent comprising a chaotropic salt having a concentration of at least 2M that denatures or degrades all proteins to produce a disrupted sample containing RNA freed from bound proteins and inactivating nucleases with no more than twenty-five percent loss of RNA; b) reducing the concentration of said chaotropic salt in said disrupted sample to less than 0.05M by diluting without washing the disrupted sample with at least one aqueous reagent before adding the reverse transcriptase; c) incubating the diluted, disrupted sample with reverse transcriptase to transcribe said RNA to cDNA; d) amplifying at least one selected sequence of said cDNA; and e) detecting the at least one amplified cDNA sequence wherein steps b) through e) are performed without first separating the RNA from degraded proteins or from said chaotropic salt.