Figure 1.
Flowchart of linear RNA amplification with the ExpressArt® mRNA Amplification Kit Nano version (AmpTech GmbH). mRNA is reverse transcribed with an anchored oligo(dT)-primer (without T7-promoter). In an attempt to minimize 3'-bias, double stranded cDNA is produced with a “Box-random-trinucleotide primer”, which preferentially binds near the 3'-ends of nucleic acid molecules. In the first round of amplification, a T7-promoter/oligo(dT) primer binds in reverse orientation to the single stranded cDNA with the Box sequence tag at the 3'end. This double stranded cDNA is then used as a template for in vitro transcription, which generates antisense oriented RNA. The antisense oriented RNA in turn is used as a template for cDNA synthesis and second round amplification.