Briefly, ExpressArt ® technology for Standard kits is based on the following steps: Conversion of mRNA to cDNA is achieved with an anchored oligo(dT) primer in the first reverse transcription reaction. No promoter sequence is present in the primary cDNAs. Then, all RNAs (including mRNAs and rRNAs) are digested with a mixture of heat-labile RNases. Single stranded cDNAs are converted to double stranded DNAs with a special primer construct, the TRinucleotide primer (BOX-random-3'-trinucleotide). This 30-mer primer contains a unique 21-mer BOX, followed by six random nucleotides, and a trinucleotide sequence at the 3'-end. We use a mix of several primers with different 3'-terminal trinucleotides.

The trinucleotides determine potential primer elongation sites, that are discontinuously distributed over annealed templates. Consequently, preferential priming near the 3'-end of the template occurs. To minimise primerderived artefacts, the T7-promotor sequence is introduced only in the 2nd synthesis of dsDNA. This double stranded cDNA is the template for the first amplification by in vitro transcription. All amplified RNAs contain the same 3'-terminal 21-mer BOX sequence. For second and third amplification rounds, full-length cDNAs are obtained using this 21-mer BOX sequence as primer. RNA removal (with heat-labile RNases) is followed by second strand cDNA synthesis, introducing a T7-promotor sequence in double stranded cDNA templates for second (or third) amplification by in vitro transcription.