ExpressArt®: The future of mRNA amplification A "NON-Eberwine" technology with unique advantages

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Use the ExpressArt® standard kits, start with Micrograms or Picograms of input total RNA

and Directly compare microarray data after One, two or three amplification rounds

Or choose from our specials: RESCUE precious samples of SEVERELY DEGRADED RNA.

SPECICIFICALLY amplify BACTERIAL mRNA.

RECOVER all mRNA sequences retained in .FFPE samples.

For ALL ExpressArt® applications we guarantee: No need to remove ribosomal RNAs, all kits are highly selective for mRNAs! No need to pre-determine input RNA amounts! Cut-off levels for your present sample processing no longer control your future! No need to discard samples which failed in standard RNA quality control

With the ExpressArt® Product Line, you can • amplify your complete sample, use picoograms or micrograms of input RNA.

• even picograms of input RNA are sufficient for multiple microarray experiments.

• directly compare microarray results with a wide range of sample sizes.

• use standard microarrays for all samples, .including degraded FFPE RNAs.. • specifically amplify bacterial mRNA from total RNA, no need to remove rRNAs.

The ExpressArt® mRNA Amplification Kits are unique and based on proprietary technology of AmpTec GmbH (patents and patents pending).

ExpressArt® kits for mRNA amplification - The Full Range in Your hands Amplified mRNA can be obtained from any eukaryotic species or from bacteria. Input total RNA can be obtained from all sample types, including laser microdissection or FACS cell sorting. Problems with severely degraded RNAs are history. – .including RNAs from FFPE samples. - when using the ExpressArt® TRinucleotide mRNA amplification kits. .Specific amplification of bacterial mRNA is reality., when using the ExpressArt® Bacterial mRNA amplification kits.

������ ��������������������� The ExpressArt® mRNA Amplification Kits provide a highly sensitive and reproducible technology for linear mRNA amplification, as well as RNA isolation, in combination with microarray hybridisation. The ExpressArt® mRNA amplification product line covers The Full Range of input total RNA: Picograms, Nanograms or Micrograms of total RNA result in sufficient amplified RNA for microarray hybridisation (low or high µg range), provided the right ExpressArt® kit version is used Use the MICRO version for input total RNA from 0.5 to 5 µg, the NANO and NANO plus versions for input total RNA from1-700 ng, the PICO version for input total RNA from 100-1000 pg,

Further, ONLY the ExpressArt® technology allows specific amplification of bacterial mRNA and complete amplification of.severely degraded mRNAs. even .from FFPE samples.

Use the TRinucleotide and FFPE mRNA amplifications kit for 5-700 ng degraded RNA or the Bacterial mRNA amplification kit for 5-700 ng bacterial RNA. Excellent reproducibility of the amplification reactions (one, two or three amplification rounds) was demonstrated by hybridisation of amplification replicates on Affymetrix GeneChips HG-U95A and HG-U133A (Pearson values at least 0.98). Qualitative and quantitative differences in the gene expression patterns are maintained, as shown in comparisons of sample pairs labelled with the standard Affymetrix protocol versus RNA samples after two amplification rounds with the ExpressArt® kit.

In addition, real-time quantitative RT-PCR experiments with mRNAs of various abundances (high, medium and low) demonstrate that amplification efficiency of the ExpressArt® mRNA Amplification Kit is approximately 1,000-fold per amplification round (�CT = 10) and is constant for genes with highly different abundances and mRNA lengths.

low copy gene medium copy gene high copy gene

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Courtesy: Baugh, Hunter at Harvard University

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Courtesy: Baugh, Hunter at Harvard University

Methodology Now, highly reproducible array hybridisations can be performed with a few cells, e.g. 4-cell embryos of C.elegans (Baugh 2004; Yanai, Baugh 2008).

Historically, linear, isothermal amplification strategy based on in vitro transcription with T7 RNA-polymerase was developed by Eberwine and coworkers (Van Gelder et al. 1990; Eberwine et al. 1992). In this procedure, mRNA was converted into double stranded cDNA, using a T7-promoter/oligo(dT) primer for first strand cDNA-synthesis and limited RNase H digestion for self-priming during second strand synthesis. For amplification, these dsDNA-molecules were used as templates for in vitro transcription. Resulting in linear amplification maintaining the expression patterns of the original mRNAs (Poirier et al. 1997; Puskas et al. 2002). A number of problems were observed with this approach: (i) amplified RNA (aRNA) is 3´-biased since transcription and cDNA-synthesis with the T7-promoter/oligo(dT) primer start at the poly(A)-tail of the original mRNA. (ii) a second amplification round is based on random priming, causing reduction of fragment length, which becomes more pronounced with ever decreasing amounts of input RNA. (iii) combination of the repetitive oligo(dT) with the T7-promotor in a primer for the promiscuous reverse transcription reaction results in high-molecular weight artefacts, which become increasingly dominant if less template is used (Baugh et al. 2001). (iv) only high quality RNA samples can be used. Eberwine technology with AmpTec ExpressArt Technology one and two amplification with two and three amplification rounds rounds Courtesy: Baugh, Hunter at Harvard University Now and for the future, the ExpressArt® mRNA Amplification Kits provide a technology, which solves these problems. With the ExpressArt® Standard kits, the mRNA is converted to cDNA with an anchored oligo(dT)-primer (but without T7-promoter). For the TRinucleotide and FFPE kits, a TRinucleotide primer is used instead. The proprietory "TRinucleotide Primer" (Box-random-3’-trinucleotide primer), results in preferential priming near the 3'-ends of all nucleic acid molecules. This is illustrated in a model experiment, using one defined, 800 nucleotides long mRNA transcript as input RNA. Only the use of TRinucleotide primers results in full-length amplified RNA, replacement of the trinucleotide by random sequence results in an array of shorter products.

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Input RNA800 nt mRNAInput RNA800 nt mRNA

Amplified RNAExpressArt TRinucleotide primerAmplified RNAExpressArt TRinucleotide primer

Amplified RNArandom primerAmplified RNArandom primer

Efficient primer elongation requires only the matching trinucleotide sequence in the template nucleic acid. This is only a minimal restriction, and the use of several primers with different trinucleotide sequences results in essentially sequence-independent universal priming. However, this minimal, but distinct sequence requirement results in efficient selection against rRNA amplification. Due to the tight secondary structures of rRNAs (and rRNA fragments!), only minimal sequence stretches are exposed, thus rarely providing the required trinucleotide sequence that is needed for TR priming. To further minimise 3´-bias in the next step, double stranded cDNA is again generated with a TRinucleotide primer and this results in the generation of almost full-length double stranded cDNAs. After denaturation, the second cDNA strand is primed in reverse orientation, using a T7-promoter/ oligo(dT) primer. This leads to double stranded cDNA with a functional T7-promotor at one end and the Box sequence tag at the other end. This dsDNA product is used as template for in vitro transcription, generating amplified, antisense oriented RNA with defined sequences at both ends. This is a major advantage for second and especially for third round amplifications, where size reductions of amplified RNAs are avoided. This is crucial and enables the comparison of samples that contain very divergent amounts of input RNA. Now, highly reproducible array hybridisations can be performed with a few cells, e.g. individual 4-cell embryos of C.elegans (Baugh 2004; Yanai, Baugh 2008). Literature Baugh LR, Hill AA, Brown EL, Hunter CP (2001). Quantitative analysis of mRNA by in vitro transcription. Nucleic

Acids Res. 29:E29 Baugh LR (2004). Genomic analysis of embryogenesis in the nematode C. elegans. Ph.D. thesis, Harvard University, Dept. Mol

Cell Biol. Boularand S, Darmon MC, Mallet J (1995). The human tryptophan hydroxylase gene: an unusual complexity in the

5'-untranslated region. J Biol Chem. 270: 3748-3756. Eberwine J, Yeh H, Miyashiro K, Cao Y, Nair S, Finell R, Zettel M, Coleman P (1992). Analysis of gene

expression in single life neurones. Proc. Natl. Acad. Sci. 89: 3010-3014. Mathieu-Daude F, Welsh J, Vogt T, McClelland M (1996). DNA rehybridization during PCR: the Cot effect and its

consequences. Nucleic Acids Res. 24: 2080-2086. Poirier F, Pyati J, Wan JS, Erlander MG (1997). Screening differentially expressed cDNA clones obtained by

differential display using amplified RNA. Nucleic Acids Res. 25: 913-914. Puskas LG, Zvara A, Hackler L, Van Hummelen P (2002). RNA amplification results in reproducible microarray

data with slight ratio bias. BioTechniques 32:1330-1340. Van Gelder RN (1990). Amplified RNA synthesised from limited quantities of heterogeneous cDNA. Proc. Natl. Acad. Sci. 87: 1663-1667. Yanai I, Baugh LR, et al. (2008). Pairing of competitive and topologically distinct regulatory modules enhances patterned gene expression. Molecular Systems Biology 4:163.

ExpressArt® 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 primer-derived 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.

total RNA

BOX

BOX

cDNA

ds cDNA

double stranded cDNA template

1 Amplificationantisense RNA

st

2 (3 ) Amplificationantisense RNA

nd rd

BOXdouble strandedcDNA template

BOX

BOX

BOX

BOX

BOX

BOX

BOX

BOX

Special I: ExpressArt® TRinucleotide mRNA amplification kits for severely degraded RNA . including

Special III: .RNA from FFPE samples..

Forgetlimitations oflow RNA quality !

The tip of the iceberg, only?

bring all your precious samples to the lightof microarray analys.

The tip of the iceberg, only?ExpressArt® TRinucleotide mRNA Amplification Kitsfor severely degraded RNAsbring all your precious samples into the light of microarray analysis

Amplification of intact and degraded mRNAs,

with and without Poly(A), while suppressing amplification

of rRNAs

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Model experiment with chemically degraded RNA

Hybridisation results with Affymetrix Genechip HG-U133AMinor loss in sensitivity (96% Relative Presence Calls)3’-5’-Ratios: GAP-DH = 2.1; ß-Actin = 0.9[ Standard Oligo(dT) primer: ß-Actin > 50 ]rRNAs not amplification: less than 2% rRNAs in aRNAs

Comparative hybridisations with Affymetrix Genechips HG-U133AScatter plot: Intact RNA (RIN = 9.5) versusseverely degraded RNA (RIN = 2.2)Pearson value, r = 0.993

Reference: intact RNA

Chemically degraded RNA

Model experiment with chemically degraded RNA

Hybridisation results with Affymetrix Genechip HG-U133AMinor loss in sensitivity (96% Relative Presence Calls)3’-5’-Ratios: GAP-DH = 2.1; ß-Actin = 0.9[ Standard Oligo(dT) primer: ß-Actin > 50 ]rRNAs not amplification: less than 2% rRNAs in aRNAs

Hybridisation results with Affymetrix Genechip HG-U133AMinor loss in sensitivity (96% Relative Presence Calls)3’-5’-Ratios: GAP-DH = 2.1; ß-Actin = 0.9[ Standard Oligo(dT) primer: ß-Actin > 50 ]rRNAs not amplification: less than 2% rRNAs in aRNAs

Comparative hybridisations with Affymetrix Genechips HG-U133AScatter plot: Intact RNA (RIN = 9.5) versusseverely degraded RNA (RIN = 2.2)Pearson value, r = 0.993

Reference: intact RNA

Chemically degraded RNA

Agilent Bioanalyzer electropherograms of 2-rounds amplified bacterial mRNA

Hybridisation results with Affymetrix E. coliGenome 2.0 GeneChips38.6% (37°C) or 46.3% (50°C) Presence Calls Signal-background ratios: 45 - 50. Scale factors of 10 to 11, Average signals (P)>4,000Suppression of rRNA amplification: less than 2% rRNAs in amplified RNAs

Agilent Bioanalyzer electropherograms of 2-rounds amplified bacterial mRNA

Hybridisation results with Affymetrix E. coliGenome 2.0 GeneChips38.6% (37°C) or 46.3% (50°C) Presence Calls Signal-background ratios: 45 - 50. Scale factors of 10 to 11, Average signals (P)>4,000Suppression of rRNA amplification: less than 2% rRNAs in amplified RNAs

Hybridisation results with Affymetrix E. coliGenome 2.0 GeneChips38.6% (37°C) or 46.3% (50°C) Presence Calls Signal-background ratios: 45 - 50. Scale factors of 10 to 11, Average signals (P)>4,000Suppression of rRNA amplification: less than 2% rRNAs in amplified RNAs

Special II: ExpressArt® Bacterial mRNA amplification kits for specific amplification of bacterial mRNA ..

Use low amounts of input RNA, 10 ng total RNA yield >50 µg amplified RNA for multiple microarray hybridisations

No need to remove bacterial rRNAs, specific mRNA amplification provides high sensitivity with high signals & high signal-to-noise ratios

Simple analysis of mixed host-pathogen samples, when working with intracellular bacteria, like Chlamydia. Use mixed bacterial & eukaryotic total RNAs, no need to remove eukaryotic rRNAs, simply remove eukaryotic Poly(A) mRNAs with Oligo(dT) beads.

Heat shock induced genes in E. coli Comparison of daa obtained with direct cDNA labelling (30 µg input RNA) versus ExpressArt® amplified mRNAs (0.1 µg input RNA)

Gene Fluorescent-

labelled cDNA

ExpressArt® amplification & fluorescent-labelled aRNA

ExpressArt® amplification & Affymetrix GeneChips

clpb medium high high dnaj medium medium medium dnak medium medium low grpe low medium medium hslj low low medium hslu low low low hslv low medium medium htpx low low medium ibpa high high high ibpb high high high lon low medium low

Amplification of bacterial mRNA or eukaryotic mRNA fragments that do not have a 3’-Poly(A): How does it work?

ExpressArt® TRinucleotide mRNA amplification technology with the following unique steps: Conversion of mRNA sequences (no PolyA necessary) to cDNA with the first TRinucleotide primer and preferential priming near the 3'-end, while preventing reverse transcription of rRNAs. Conversion of cDNAs to double stranded DNAs with a second TRinucleotide primer. All amplified RNAs contain a 5' Box-1 and a 3' BOX-2 sequence, and simply use the BOX-2 primer for second and third amplification rounds, rendering them highly reliable.

mRNAs or mRNA fragmentswithout Poly(A)

BOX-2

BOX-2

cDNA

ds cDNA

double stranded cDNA template

1 Amplificationantisense RNA

st

2 Amplificationantisense RNA

nd

BOXdouble strandedcDNA template

BOX-2

BOX

BOX-2

BOX

BOX-2

BOX

BOX-2

BOX

Key Advantages of this New mRNA Amplification Technology 1. Special kits for specific amplification of bacterial mRNA

2. Special kits for amplification of severely degraded RNA including RNA from FFPE samples selective mRNA amplification and full sequence recovery

3. No primer derived artefacts cDNA synthesis is uncoupled from insertion of T7-promotor With other systems, the frequently observed large amounts of template-independent high molecular weight amplification artefacts are a severe limitation in the amplification of very low amounts of input RNA. With ExpressArt®, the “no-template-control“ is free of any amplified background, even after two and three rounds of amplification. This enables the amplification of sub-nanogram amounts of input total RNA, as demonstrated by the amplification of RNA from 4-cell embryos of C. elegans (Baugh, Ph.D. thesis, Harvard University, 2004; Yanai, Baugh 2008).

4. Amplified RNAs contain defined sequences at both ends

In addition to the 5'-proximal oligo(U) stretch, all amplified RNAs contain a unique 3'-terminal Box sequence.

5. No continuous shortening with loss of mRNA sequences dsDNA with "TRinucleotide primer" (Box-random-3’-trinucleotide primer) not with random primers

Three amplification rounds as faithful as two. Full comparability is obtained.

6. Optional second or third amplification round guarantees: Absolutely unique flexibility Flexible transition between one, two or three rounds, between laser microdissection, cryosections, biopsies etc. No need for careful control of input RNA amounts. Small and large amounts can be directly compared, regardless if they require one, or two, or three amplification rounds. Rescue of drop-outs in series with one or two amplification rounds. An additional second or third round can be performed, but required only for those samples with insufficient yields in the previous round. 2-rounds (100ng) vs. 2-rounds (10ng) 2-rounds (100ng) vs 3-rounds (5ng) Competitors claim it,

ExpressArt® has achieved it: r=0.994

r=0.987

Good concordance of microarray data with one and two amplification rounds r=0.963

1-round (2µg) vs. 2-rounds (10ng)

7. Archival templates (AT versions of all kits available) Simple and easy multiple recovery of amplified RNAs, for re-evaluation of old samples in new projects, e.g. with changed microarray designs. Template (transcription reaction in solution) Archival Template (fresh; solid phase) vs Archival Template (solid phase transcription) vs. Archival Template (stored; solid phase )

r=0.994 r=0.992 8. Faithful reproduction of dynamic gene expression levels, even comparing fresh/frozen (...) and FFPE (...) samples Correct assignment of gene expression changes, preserved fold changes, from low to high levels.

Reference Users (in alphabetical order) John Arrand, Institute for Cancer Studies, University of Birmingham, U.K.

Karl Baumforth, Institute for Cancer Studies, University of Birmingham, U.K.

Ryan Baugh (now CalTech) & Craig Hunter, Harvard University, USA

Bodo Brunner, Aventis Pharma GmbH, Group aventis-sanofi, Germany

Ludger Klein-Hitpass, Array Facility, University Hospital, Essen, Germany*1

Veska Uzunov, Novartis Pharma AG, Basel, Switzerland

Ulrich Sauer, PALM Microlaser Technologies, Bernried, Germany*2

André Imhof, MMI, Molecular Machines & Industries, Glattbrugg, Switzerland*3

Rosemarie Walter, Asterand plc, Detroit, USA

David Wong, UCLA, Institute for Dentistry, Los Angeles, USA

* Application Notes available *1 RNA from FACS sorted stem cells *2 Laser Microdissection (PALM), RNA isolation, RNA amplification, microarray analysis *3 Laser Microdissection (MMI), RNA isolation, RNA amplification, microarray analysis ** Special: Stringent RNA quality control with Agilent 2100 bioanalyzer

Discordant signals: ~ 1% - This means, of 100 differentially expressed genes, one could be discordant between pairs of fresh/frozen versus FFPE samples.

Expected yields and length distribution of amplified RNAs Please note that mRNA content varies with sample type, ranging between 1 and 5% of total RNA. Data presented were obtained with total RNA, isolated from cultured cells (about 3% mRNA).

C&E Version Pico kit Input total RNA 1st aRNA 2nd aRNA 3rd aRNA 100 pg not applicable 2 to 10 µg > 50 µg 1 ng not applicable 20 ± 10 µg > 50 µg

C&E Version Nano kit <1 ng not applicable 20 ± 10 µg > 50 µg 50 ng 3 ± 0.5 µg > 50 µg not applicable 100 ng 5 ± 1 µg > 50 µg not applicable

C&E Version Micro kit - only one amplification round -

Input total RNA Yield (4 h in vitro transcription) Yield (16 h in vitro transcription) 200 ng 5 ± 2 µg 10 ± 2 µg 300 ng 10 ± 4 µg 15 ± 4 µg 1 µg 50 ± 10 µg 50 ± 10 µg

aa Example Electropherograms of ExpressArt® amplified mRNAs RNA profiles were obtained with the Agilent Bioanalyzer Input amount: ~ 10 nanogram total RNA (high quality)

1st round amplified RNA 2nd round amplified RNA 3rd round amplified RNA

Input amount: ~ 200 picogram Input amount: total RNA (high quality) .~ 50 ng total RNA (FFPE sample)..

2nd round amplified RNA 2nd round amplified RNA: 23% Presence Calls on Affymetrix HG-U133v2.0

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Ordering Information ExpressArt® Product Line - The Full Range in your Hands

The Standard mRNA Amplification Kits for Good Quality Eukaryotic RNA Input total RNA ExpressArt® C&E mRNA amplification kits Cat. No

0.3 – 3µg C&E MICRO kit for 30x 1-round (30 reactions) 7199-A30 1 – 700ng C&E NANO kit for 15x 2-rounds (30 reactions) 7299-A15

100 – 1.000pg C&E PICO kit for 15x 3-rounds (45 reactions) 7399-A15 Special I ExpressArt® TRinucleotide mRNA Amplification Kits

for degraded RNA & FFPE RNA. suitable for whole transcript (Exon or Gene ST) arrays

0.3 – 3µg C&E TR MICRO kit for 30x 1-round (30 reactions) 6199-A30 1 – 700ng C&E TR NANO kit for 15x 2-rounds (30 reactions) 6299-A15

100 – 1.000pg C&E TR PICO kit for 15x 3-rounds (45 reactions) 6399-A15 Special II ExpressArt® .Bacterial mRNA. Amplification Kits

0.3 – 3µg C&E Bacterial MICRO kit for 30x 1-round (30 reactions) 5199-A30 1 – 700ng C&E TR NANO kit for 15x 2-rounds (30 reactions) 5299-A15

No need to remove rRNAs, TRinucleotide priming enables preferential mRNA amplification

ExpressArt® Add-On Modules 2000-A30 for 30 rxns

AminoAllyl Add-On Module

Generation of AminoAllyl modified RNAs All reagents are provided,

including dye coupling and RNA fragmentation buffers, but excluding NHS-activated dyes

2000-A15 for 15 rxns 2010-A30 for 30 rxns

Archival Template Add-On Module

Generation of Immobilised Template DNAs These Archival Templates can be re-used immediately

after test with unmodified RNA, to generate labelled RNAs; or after months of storage (4°C) with novel microarrays or

in a different experimental context.

2010-A15 for 15 rxns

ExpressArt® Reagents Pico RNA

Care Carrier compounds for very small samples (< 10ng RNA)

N-Carrier (RNA) and P-Carrier (carrier for EtOH precipitations)

8999-A100 for 100 samples

NucleoGuard (NG)

Universal nuclease and RNase inhibitor for improved RNA quality, especially for laser

microdissection acts as nucleic acid mimic in resolving high molecular weight aggregates in FFPE RNAs to improve template performance

8998-M50 for 50 ml

lysate

DeCrossLinker (DCL)

FFPE RNA Enhance Combination of NG and DCL (reversal of FFPE cross-links)

resolves high molecular weight aggregates and results in improved template performance

8990-M50 for 50 ml

lysate

For further information please contact

www.amp-tec.com

Dr. Guido Krupp | krupp@amp-tec.com

Dr. Peter Scheinert | scheinert@amp-tec.com