Methods and compositions for the amplification of mRNA

An in vitro method for preparing antigen-primed human dendritic cells for use in eliciting an immune response, the method comprising

• a) preparing an RNA by transcribing a cDNA synthesized from an mRNA, the RNA prepared by a method comprising:
• i) in an in vitro reaction mixture, hybridizing a first primer to the mRNA, wherein the first primer comprises both SEQ ID NO:
• 2 and a poly(T) sequence whereby the first primer anneals to the mRNA;
• ii) extending the first primer with a reverse transcriptase that has terminal transferase and template switching activity to generate a partial first cDNA strand with an oligo(C) overhang;
• iii) hybridizing a second primer comprising SEQ ID NO:
• 1 to the oligo(C) overhang of the partial first cDNA strand;
• iv) extending the partial first cDNA strand in the 3′ direction from the oligo(C) overhang using the second primer as a template, thereby generating a first cDNA strand;
• v) hybridizing a third primer that comprises an RNA promoter to the first cDNA strand;
• vi) extending the third primer to generate a second cDNA strand, thereby generating a double stranded cDNA;
• and
• vii) synthesizing RNA from the double stranded cDNA;
• and
• b) priming the human dendritic cell with the synthesized RNA to provide the antigen-primed human dendritic cells for use in eliciting an immune response.