A random-primed reverse transcriptase-in vitro transcription method of
linearly amplifying RNA is provided. According to the methods of the
invention, source RNA (or other single-stranded nucleic acid),
preferably, mRNA, is converted to double-stranded cDNA using two random
primers, one of which comprises a RNA polymerase promoter sequence
("promoter-primer"), to yield a double-stranded cDNA that comprises a RNA
polymerase promoter that is recognized by a RNA polymerase. Preferably,
the primer for first-strand cDNA synthesis is a promoter-primer and the
primer for second-strand cDNA synthesis is not a promoter-primer. The
double-stranded cDNA is then transcribed into RNA by the RNA polymerase,
optimally in the presence of a reverse transcriptase that is rendered
incapable of RNA-dependent DNA polymerase activity during this
transcription step. The subject methods produce linearly amplified RNA
with little or no 3' bias in the sequences of the nucleic acid population
amplified.