AmpTec Amplification Technologies

ExpressArt® mRNA Amplification includes the following steps:

Standard mRNA amplification Kits

Amplification target is poly(A) mRNA and the starting material is intact eukaryotic total RNA. 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 (5'-BOX-random-trinucleo- tide-3'). This 30mer primer contains a unique 21mer 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 primer derived artefacts, the T7-promotor sequence is introduced only in the 2^nd 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 21mer BOX sequence.

For second and third amplification rounds, full-length cDNAs are obtained using this 21mer 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.

 

Special mRNA amplification Kits

Amplification targets are intact or degraded mRNAs, with or without 3'-poly(A), and the starting material is degraded eukaryotic total RNA or bacterial total RNA. Conversion of mRNA to cDNA is achieved with the first TRinucleotide primer. This special 30mer primer construct (5'-BOX-1-random-trinucleotide-3') contains a unique 21mer BOX 1, 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, whereas reverse transcription of rRNAs is prevented. 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 the second TRinucleotide primer (5'-BOX-2-random-trinucleotide-3'). To minimise primer derived artefacts, the T7-promotor sequence is introduced only in the 2^nd synthesis of dsDNA. This double stranded cDNA is the template for the first amplification by in vitro transcription. All amplified RNAs contain the same 5'-terminal BOX 1 and 3'-terminal BOX 2 sequence.

For second and third amplification rounds, full-length cDNAs are obtained using the 21mer BOX 2 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.