AmpTec Amplification Technologies

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ExpressArt® - Frequently Asked Questions

Topic Question Answer(s)
General Advantages 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).

Which publications have used ExpressArt products and ExpressArt technology? Pico RNA Care:
Campean et al., 2008. Am J Physiol Renal Physiol 294: F1174-84.
Su et al., 2008. J Immunol 181:1264â€1271.
Wetzel et al., 2006. Kidney Int. 70: 717-723.
Zeng et al., 2005. EMBO J. 23: 4116-4125.

NucleoGuard:
Hu et al., 2008. Clin.Chem. 54:824-832

mRNA Amplification Kits:
Baumforth et al., 2008. Am. J. Pathol. 173:195-204.
Hu et al., 2008. Clinical Chemistry 54(5): 824-832.
Yanai, Baugh et al., 2008. Mol. Syst. Biol. 4:163.
Cummings et al., 2008. Proc. R. Soc. B 275: 393-402.
Duftner et al., 2008. Genomics 91: 108-117.
Birgersdotter et al., 2007. Leuk. Lymph. 48: 2042-2053.
Bose et al., 2007. J. Pathol. 213:329-336.
Eickhoff et al., 2007. Infect. Immun. 75: 2853-2863.
McLucas et al., 2006. J. Biomed. Mater. Res. A: 246-253.
Reis et al., 2006. J. Mol. Histol. 37: 79-86.
Okuducu et al., 2005. Int. J. Oncol. 27: 1273-1282.
Laser Microdissection What is the recommended method for RNA isolation and for storage and for shipping of isolated RNAs? Standard RNA isolation kits can be used, like RNeasy from Qiagen or PicoPure from Arcturus (now MDS Pharma Service), but they should be supplemented with ExpressArt N-Carrier (#8999-A100) and ExpressArt NucleoGuard (#8998-M50).
Storage & shipping: Add salt (NaAc) and P-Carrier (included with ExpressArt RNA Care #8999-A100) and EtOH. This mixture (an RNA suspension) can be stored at -20°C or shipped with CoolPacks or on dry ice.
RNA is recovered by centrifugation and inclusion of the P-Carrier results in a visible pink pellet and it further ensures reliable RNA recovery, even in the picogram range.

What are the benefits of "N-Carrier" and of "NucleoGuard"? N-Carrier is an RNA with a hairpin structure that makes it totally inert (no primer, no template). Its small size ensures effective removal (>99%) by spin columns and prevents any interference with Agilent RNA profiles (even with picogram RNA isolates).
NucleoGuard is an effective and universal nuclease inhibitor and its chemical nature ensures full activity even in high GTC salt concentrations. Although GTC in common lysis buffers is a denaturing agent, not all enzymes (RNases) are completely inactivated, as is obvious in the use of proteinase K in GTC buffers.
Benefit of NucleoGuard is shown in a recent publication:
Hu et al. (2008) Clin.Chem. 54:824-832. Supplementary Figure 2 demonstrates recovery of rRNA peaks - but only if NucleoGuard had been included in the lysis step.

Which publications have used ExpressArt for Laser Microdis-sected samples? - or have demonstrated the benefits of NG? Pico RNA Care:
Campean et al., 2008. Am J Physiol Renal Physiol 294: F1174-84.
Su et al., 2008. J Immunol 181:1264â€1271.
Wetzel et al., 2006. Kidney Int. 70: 717-723.
Zeng et al., 2005. EMBO J. 23: 4116-4125.

NucleoGuard:
Hu et al., 2008. Clin.Chem. 54:824-832

mRNA Amplification Kits:
Baumforth et al., 2008. Am. J. Pathol. 173:195-204.
Birgersdotter et al., 2007. Leuk. Lymph. 48: 2042-2053.
Bose et al., 2007. J. Pathol. 213:329-336.
Reis et al., 2006. J. Mol. Histol. 37: 79-86.
Okuducu et al., 2005. Int. J. Oncol. 27: 1273-1282.

Bacterial RNA samples What happens with mRNAs without the universal 3' poly-A? The patented ExpressArt TRinucleotide priming technology results in preferential priming near the 3'-ends of mRNAs (similar effect as oligo-dT with poly-A).

What happens with ribosomal RNA? - And do I have to check if my bacterial species is in a recommendation list? The patented ExpressArt TRinucleotide priming technology selects for mRNAs and against rRNAs. This selection is based on the compact rRNA structure. This is required for biological rRNA function; therefore it is a universal feature and there is no need to check lists.

What can I do if there is no microarray for "my species"? Combimatrix offers highly flexible microarray design at low cost and with short turn-around times; see: http://www.combimatrix.com/support_faq.htm.

Degraded RNA samples What happens with mRNA fragments without the 3' poly-A? The patented ExpressArt TRinucleotide priming technology results in preferential priming near the 3'-ends of mRNAs (similar effect as oligo-dT with poly-A).

What happens with ribosomal RNA? The patented ExpressArt TRinucleotide priming technology selects for mRNAs and against rRNAs. This selection is based on the compact rRNA structure. This is required for biological rRNA function; therefore it is a universal feature of all rRNAs and degraded rRNA fragments are even enriched in compact structures.

Ribosomal RNA What is the principle for selection against rRNA amplification? -And do I have to check if my species is in a recommendation list? The patented ExpressArt TRinucleotide priming technology selects for mRNAs and against rRNAs. This selection is based on the compact rRNA structure. This is required for biological rRNA function; therefore it is a universal feature and there is no need to check lists.
For primer elongation with TRinucleotide primers, there is a low, but distinct sequence requirement for a small set of defined trinucleotide sequences in the template. Since most sequences in rRNAs are hidden in compact sections, the short exposed sequence regions rarely contain these required trinucleotide sequences, and there is no effective primer elongation.

What are the advantages? Amplified RNAs have a low percentage of rRNA sequences (<2% in 2-rounds amplified RNAs). In microarray hybridisations, rRNAs result in high background signals and reduced detection sensitivity. With ExpressArt amplified RNAs superior Signal/Background ratios are obtained.

Paraffin (FFPE) samples What is the recommended method for RNA isolation? Your choice of a commercial FFPE in combination with ExpressArt FFPE RNA Enhance.

What are the benefits of FFPE RNA Enhance (DeCrossLinker" plus "NucleoGuard")? ExpressArt Additives result in better FFPE RNA quality: size distribution is improved with longer mRNA fragments and on the other hand, high-molecular-weight RNA aggregates are resolved, in effect the RNA template quality is improved with lower ΔCt-values in qPCR assays (~ 3 cycles).

What is special for ExpressArt FFPE mRNA amplification? Different to random priming with other kits, the TRinucleotide technology results in preferred 3'-proximal priming and in low percentage of rRNA sequences (<2%). This combination results in better mRNA sequence recovery and better Signal/Background ratios.

Whole Transcript Arrays: Exon or Gene ST Arrays These whole transcript arrays require amplification of all mRNA sequences, not only of a short stretch (near the poly-A). What is so special for ExpressArt in these applications? ExpressArt is easier and faster.
The rRNA removal step is avoided and due to the very low percentage of rRNA sequences (<2%), the sensitivity with ExpressArt amplified is superior.

Why can I use very low RNA amounts (<100 ng total RNA) only with ExpressArt? All other kits use random priming with high amplification yields for rRNAs which leads to reduced sensitivities: lower number of detected genes. This can be avoided by an extra step for (partial: ~70%) removal of rRNAs. This step uses magnetic beads which require an absolute minimum of 1 μg of total RNA, otherwise all RNA will be lost during the procedure.
All other kits offer only one amplification round.
ExpressArt avoids random priming: the patented TRinucleotide priming selects against rRNAs (~98% removal) and ExpressArt offers two (even three) amplification rounds.

Why can I use degraded RNAs only with ExpressArt? All other kits require rRNA removal for optimal sensitivity. This step uses rRNA-specific, biotinylated oligos that hybridise with rRNAs and the hybrids are captured by magnetic beads. If rRNAs are degraded, only the rRNA segment(s) containing the target sequence can be removed, all other rRNA sequences remain in the sample and are amplified by random priming.
ExpressArt amplification is based on selection against compact rRNA structures, and rRNA fragments tend to be even more compact than intact rRNAs, and ~98% removal of rRNA sequences is maintained also with degraded RNA.

Which publication has used ExpressArt for whole transcript arrays? Hu et al. (2008) Clin.Chem. 54:824-832.
Affymetrix GeneChip Human Exon 1.0 ST Arrays with very small (1-10 ng) and severely degraded total RNA samples from saliva (Bioanalyzer RIN is 2.3-3.2, see Supplement, Fig. 3).

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