Application Info

Amp_Tec_logoExpressArt® mRNA amplification technology for intact RNA: How does it work?

 

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.

 

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RNA isolations from archival paraffin samples adopted for QuickGene-Mini80!quickgene_mini80

Concentrated RNA product that is ready-to-use for qRT-PCR, mRNA amplification and microarray analyses.

 No overnight lysis step required !  - No centrifugation steps required !

 

What is so special about ExpressArt technology? 

No calibrated amounts are needed.
Whereas all other kits demand for very similar RNA amounts in sample series. ExpressArt avoids the size reductions of other kits (less input RNA gives shorter amplified RNA). Therefore, ExpressArt provides excellent comparability for different sample sizes: RNA from a few cells or from hundreds or thousands of cells obtained from
Laser microdissection, cell sorting, isolated embryos or oocytes.

Amplification of Picogram samples without large primer-derived artefacts.
Combined with the avoided size reductions, this gives excellent results with picograms of input RNA.
Unique ability to use three amplification rounds - without any compromise in data quality and with high concordance in direct comparisons of two and three amplification rounds.

No dead-end with labelled amplified RNAs.
Use Archival Template DNA (#2010-A15), immobilised on magnetic beads, for solid-phase in vitro transcription with unmodified NTPs.
Only if quantitity and quality of the amplified RNA are O.K., simply recover the template DNA and repeat the in vitro transcription reaction with labelled NTP's. Otherwise, use the unmodified RNA to perform an additional amplification round.

No standard RNA quality is needed.
ExpressArt avoids random priming and the patented TRinucleotide priming technology gives highly comparable results in sample series with perfect RNA quality (Agilent RIN>9), reduced quality (RIN 6-9) or low quality (RIN<6).

ExpressArt® - Frequently Asked Questions >>