A novel approach to selectively amplify mRNA in challenging samples

“ExpressArt”

Dr. Guido KruppAmpTec

Amplification Technologies

AmpTecAmplification Technologies

The University of VermontBurlingtonAugust 22, 2008

The ExpressArt Technology

FIRST → TRinucleotide priming versus conventional Eberwine technology

for small and divergent sample sizes

• Microarrays and qRT-PCR

• Laser Microdissection, FACS-sorted cells

• “Helpers“ for RNA recovery

SECOND → TRinucleotide priming for reverse transcription: intact and degraded RNAs

with selection for mRNAs and against rRNAs

• FFPE samples with “Helpers“ for RNA recovery

• Exon Arrays: Poor RNA quality with very small samples

THIRD → TRinucleotide priming for reverse transcription: bacterial RNAs

with selection for mRNAs and against rRNAs

• Selective lysis of intracellular bacteria

Linear amplificationby in vitro transcription

Mix 1 2 3 4 M M Mix 1 2 3 4

Exponential, cyclic amplificationper PCR

Mix 1 2 3 4 M Mix 1 2 3 4 M

Random Variability1% per template generationThousandfold in 10 cycles: 0.9910 or 0.90Millionfold in 20 cycles: 0.9920 or 0.82

Random Variability1% per template generationThousandfold in 1-round: 0.99 Millionfold in 2-rounds: 0.992 or 0.9801

Amplification of mixed DNA templates with equimolar amounts

• Size matters • Abundance matters

Why linear amplification?

mRNA amplification technologiesProblems with very small samples

AmpTec ExpressArt® Technology*with two and three amplification rounds

* Courtesy: Baugh & Hunter, Harvard University

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“Eberwine Method” with one and two amplification rounds

M

Eberwine: problems with high molecular weight artefacts

Eberwine method

Mike Mallamaci, AstraZeneca

ExpressArt® Technology

2-rounds amplified RNA

3-rounds amplified RNA

Assessing cRNA Qualities2 Amplification rounds, 2 ng input RNA

18S rRNA position as marker

T7

T7

Eberwine Scheme I: Synthesis of DNA template for T7 RNA Polymerase

RNA fragments as primers in second strand cDNA synthesis

Limited RNase H digestion

Reverse transcription of mRNAWith Oligo(dT)-T7 primer

Eberwine Scheme II: Transcription for first mRNA amplification round

T7

Eberwine Scheme III: Second round synthesis of DNA templateProblems with divergent sample sizes

3´-BIAS: Functional T7 promotor only in 3´-terminal cDNA templates

T7

Random primers in cDNA synthesisHigh amount of template

Low primer excess

AAAAA 3´

AAAAA TTTTT

Low amount of template High primer excess

AAAAA 3´

T7AAAAA TTTTT

Short antisense RNAspreferrentially representing the 3‘-proximal section of mRNAs

T7

Eberwine Scheme IV: Transcription for second mRNA amplification round

ds DNA with TRinucleotide-Primer-Mix Including a 5‘ Box sequence

T T T T T -5’

B BT T T T T -5’ T T T T T -5’A A A A A -3’ A A A A A -3’

“Full-length“ products

ExpressArt Scheme I: Synthesis of ds cDNA

ExpressArt Scheme II: Function of TRinucleotide Primers

Box

TCTB

ox

TCT

Box

TCT

Box

TCTTTTTT-5’AGA AGA AGA AGA

Box

TTTTT-5’AGATCT

AGA AGA AGAAAAAA-3’

21-mer Box + 6-mer Random + Trinucleotide Mix

ExpressArt: Amplification of a model transcript

Input RNA800 nt mRNA

Amplified RNAExpressArt TRinucleotide primer

ExpressArt: Amplification of a model transcript

Amplified RNAExpressArt TRinucleotide primer

Amplified RNArandom primer

B

B

B

B

BB

B

U U U U U -5 ’

U U U U U -5 ’

U U U U U -5 ’

U U U U U -5 ’

in vitro transcription

dsDNA with oligo(dT)-T7 primer

T T T T T -5’

T T T T T

A A A A A -3’

A A A A A -3’

A A A A A T7

ExpressArt Scheme III:Synthesis of DNA template and transcription for first round

First AMPLIFICATION

BU U U U U -5 ’

1) Reverse transcription of amplified RNAwith

2) RNase to destroy RNA

3) dsDNA with Oligo(dT)-T7

Box-Primer

-Primer

in vitro transcription

B

BB

B

U U U U U -5 ’

U U U U U -5 ’

U U U U U -5 ’

U U U U U -5 ’

BT T T T TA A A A A T7

ExpressArt Scheme IV:DNA template and transcription for second Round

Second AMPLIFICATION

ExpressArt Results: Reproducibility & Comparability

High concordance of microarray data with divergent sample sizes

r=0.994

2 rounds: 100ng versus 1.5ng

ExpressArt® Eberwine*

r=0.935*

2 rounds: 50ng versus 10ng

PCR-coupled amplification*

r=0.979*

Roche Kit: PCR + IVT 50ng versus 10ng

*Reference: Klur et al. (2004) Genomics 83, 508-517

Evaluation of ExpressArt mRNA amplification kits

starting with

high (4 µg) and very low amounts (2ng) of input total RNA

& comparing one, two and three amplification rounds

Using high quality mouse liver and kidney total RNAs in triplicate experiments

* * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *

Bodo Brunner, Ph.D.

Group sanofi-aventis

Aventis Pharma Deutschland GmbH

Mean foldchange ExpressArt 20ng 2x IVT

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r = 0.983

Global foldchange analysis kidney vs. liver (4)

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ExpressArt kit for mRNA AmplificationPico-version

Ryan Baugh, Kate Hill-Harfe, Gene Brown and Craig Hunter

Dept. of Molecular and Cellular Biology, Harvard UniversityExpression Profiling Sciences, Wyeth Research25 June 2003

The nematode Caenorhabditis elegans

4-cell-embryo

Genomic Analysis of Embryonic Gene Expression in C. elegans

Ryan Baugh, Ph.D. Thesis, Harvard, 2003Yanai, Baugh et al. (2008) Mol. Syst. Biol. 4:163

Descriptive Statistics

Standard_1 Standard_2 Baugh_2ng_1 Baugh_2ng_2 Pico_1 Pico_2Average Signal 19,5 18,4 29,2 16,9 20,8 20,0Median Signal 2 2 4 2 2 3Max Signal 1777 1538 1615 1129 2312 1737Present Calls 2964 2926 3373 2995 3639 3636

44.8% 44.2% 50.9% 44.7% 55.0% 54.9%

 

 

Standard 10µg total RNA from Jurkat & H9 cellshybridization with HG‑U133A

Presence calls: 9516 (42.8%)

2‑rounds ExpressArt 100ng total RNA from Jurkat & H9 cellshybridization with HG‑U133A

Presence calls: 10149 (45.7%)

Additional genes: 633 Median mRNA length: 4100 nucleotides

Total number of genes on C.elegans Affymetrix Gene Chip: 6617

Heat map of correlation matrix

5x 10-embryosBaugh5x 10-embryosBaugh

3x 1-embryo(4-cells)

2x 10-embryos

3x 1 embryo(4-cells)

2x 10 embryos

1

Yanai, Baugh et al. (2008) Pairing of competitive and topologically distinctregulatory modules enhances patterned gene expression. Molecular Systems Biology 4: 163

“We used RNAi and time series, whole-genome microarray analyses tosystematically perturb and characterize components of a Caenorhabditiselegans lineage-specific transcriptional regulatory network.”

Heat your samples: 2 min @ 70°C

Watch out for your carrier!

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RNA with heating step

Total RNA – microdissected sample with 300 mouse liver cells

-- a 10% aliquot: appr. 0.3 ng

-- ExpressArt N-carrier added: 100 ng (initially)

First “Helper“ for RNA recovery: N-Carrier

Second “Helper“ for RNA recovery: NucleoGuard

“The Wong Lab At the UCLA School of Dentistry“Hu et al., 2008Exon-level expression profiling: a comprehensive transcriptome analysis for oral fluids. Clinical Chemistry 54:5.

RNA profiles: blue without red with NucleoGuard

Dr. Rosemarie Walter Research Lab at Asterand plc, Detroit, USA

RNA profiles: blue without red with NucleoGuard

Second “Helper“ for RNA recovery: NucleoGuard

S t o i n s k i - B a n g s e t a l . 2 0 0 5 A P P L I C A T I O N N O T E a r t u s G m b H - M M I A G

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C e l l C u t ® L A S E R M I C R O D I S S E C T I O N

M M I A Gw w w . m m i - m i c r o . c o m

E x p r e s s A r t ® m R N A A M P L I F I C A T I O N

a r t u s G m b Hw w w . a r t u s - b i o t e c h . c o m

S t o i n s k i - B a n g s e t a l . 2 0 0 5 A P P L I C A T I O N N O T E a r t u s G m b H - M M I A G

“Helpers“ for LCM samplesCampean et al., 2008. Am J Physiol Renal Physiol 294: F1174-84.Su et al., 2008. J Immunol 181:1264–1271.Wetzel et al., 2006. Kidney Int. 70: 717-723.Users: Novartis, Basel, Switzerland Asterand, Detroit, USA“Helpers“ for FACS-sorted cellsZeng et al., 2005. EMBO J. 23: 4116-4125.

Amplification Kits for LCM samplesBaumforth 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.

Amplification Kits for FACS-sorted cells”germinal centre (GC) B cells”Vockerodt et al., 2008. J. Pathol. 216:83–92.

PAX5

18428 18403 18376 18425 184310

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Differential gene expression by qRT-PCR analysisComparison of non-amplified total RNA and ExpressArt amplified RNA (2 rounds)

Dr. Monika SczepanowskiCentre for Applied Cancer ResearchUniversity Clinic Schleswig-Holstein, Kiel, Germany

SECOND → TRinucleotide priming for reverse transcription

Model system for degraded RNA

AAAAAA

AAAAAA

A

Controlled cleavage without loss of sequence information

Is it possible to recover the complete sequence informations from all fragments?

Fragmentation reactionMetal-catalyzed cleavage at elevated pH & temperature

Function of Box-Random-TRinucleotide-Primer-Mix

Box

TCTB

ox

TCT

Box

TCT

Box

TCTTTTTT-5’AGA AGA AGA AGA

Box

TTTTT-5’AGATCT

AGA AGA AGAAAAAA-3’

AAAAAA

AFirst cDNA synthesisPreferential priming near the 3’-end of any nucleic acid sequence

Second cDNA synthesisPreferential priming near the 3’-end

AAAAAA

A

BoxBox

BoxBox

BoxBox

BoxBox2

Third DNA synthesisBox-T7-promotor primer

BoxBox2 T7

Box

RNA removal

BoxBox2 T7

In vitro transcription & amplification

BoxBox

Box

Box

Box2Box2

Box2

Box2

Amplified antisense RNA

Reverse transcription with Box2 primerRNA removalDNA synthesis with Box-T7-promotor primer

SecondRound

BoxBox2 T7

RIN = 3.8

RIN = 3.1

RIN = 2.2

RIN = 9.5 Hybridisation Results Affymetrix GeneChips HG-U133A

Presence Calls 3’-5’-ratios rRNAs [%]GAP-DH ß-actin

9,794 [:= 100%] 3.2 1.6 1.8%

98% 2.8 1.1 2%

98% 2.0 0.9 1.9%

96% 2.5 0.9 1.8%

cDNA with Oligo(dT)82% >15 >50 0.4% M:absent 5’:absent

TRinucleotide mRNA AmplificationIntact versus Severely Degraded RNAr = 0.993

TRinucleotide mRNA AmplificationIntact versus Severely Degraded RNAr = 0.993

Standard mRNA amplification with Oligo(dT)

r = 0.793

Compact Structure of ribosomal RNAs

ExpressArt® - The future of mRNA amplification

&High-Quality Microarrays

RNA from FFPE Samples

“Helpers“ for FFPE RNA recoveryDCL (DeCrossLinker) & NG (NucleoGuard)

No additives

Lysis in presence of DCL & NG

Useful as template in RT-qPCR: Ct = 4 – 10

Dr. Rosemarie Walter Research Lab at Asterand plc, Detroit, USA

Standard Procedure(Affymetrix)

Starting MaterialMinimum of 1-2 µg total RNA

Intact ONLY

rRNA depletion

Enriched mRNA300-600 ng

1st double strandedDNA synthesis

double strandedtemplate DNA

Micro Procedure(AmpTec)

1st double strandedDNA synthesisTRinucleotide Priming isSelective against rRNA

double strandedtemplate DNA

Starting MaterialMinimum of 5 ng total RNA

Intact OR Degraded

1st double strandedDNA synthesisTRinucleotide Priming isSelective against rRNA

1st double strandedtemplate DNA

Nano Procedure(AmpTec)

1st IVT Amplification withT7 RNA polymerase

1st cRNA

2nd double strandedDNA synthesis

2nd double strandedtemplate DNA

> 7 µg cRNA

2nd Cycle Double StrandedcDNA synthesis

> 7.5 µg dsDNA (with dUTP)

Fragmentation &Labelling

Fragmented / Labelled DNA

Hybridisation to Affymetrix Exon ST Arrays or Gene ST Arrays

GeneChip® WT Double-Stranded cDNA Synthesis Kit

GeneChip® WT Terminal Labeling Kit

Starting MaterialMinimum of 0.5 µg total RNA

Intact OR Degraded

IVT Amplification withT7 RNA polymerase

Whole Transcript ST ArraysmRNA Amplification without rRNA Depletion

EXON ARRAYS

Quality of Input RNA Samples and Overview of Hybridisation Results

Model experiment. Intact RNA samples were chemically degraded, to maintain full sequence complexity in degraded RNA samples

Intact RNAIntact RNAIntact RNAIntact RNA Degraded RNA“1000 nt“Degraded RNA“1000 nt“Degraded RNA“1000 nt“Degraded RNA“1000 nt“ Severely Degraded RNA

“500 nt“Severely Degraded RNA“500 nt“

Severely Degraded RNA“500 nt“

Severely Degraded RNA“500 nt“Severely Degraded RNA“500 nt“Severely Degraded RNA“500 nt“Severely Degraded RNA“500 nt“

Severely Degraded RNA“500 nt“

Method for generating cRNAs

Starting material RNA amount / RNA quality

cRNA yields [µg]

Sensitivity [% P]

Mean Signal vs Background (Ratio)

Biological replicates [Pearson values]

ROC pos-neg cont

Standard Affymetrix

2.0 µg / intact 21 ± 5 51 ± 1 280 vs 310 (0.9)

0.98 0.881 ± 0.003

AmpTec TRinucleotide

50 ng / intact 2 rounds* 62 ± 10

64 ± 2 360 vs 210 (1.7)

0.98 0.901 ± 0.004

AmpTec TRinucleotide

50 ng / degraded ("1000nt")

2 rounds* 58 ± 10

53 ± 2 280 vs 200 (1.5)

0.96 0.892 ± 0.004

AmpTec TRinucleotide

50 ng / severely degraded ("500nt")

2 rounds* 52 ± 5

47 ± 3 265 vs 250 (1.1)

0.95 0.875 ± 0.003

Overview of Hybridisation Results

Signal Intensities for Exon Probes over the Complete Length of GAP-DH mRNA

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Sig

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TR Intact

TR Degraded

TR 75" Degraded

Extreme Samples: Low amounts (< 4 ng) of severely degraded saliva RNA“The Wong Lab At the UCLA School of Dentistry“Hu et al., 2008Exon-level expression profiling: a comprehensive transcriptome analysis for oral fluids. Clinical Chemistry 54:5.

Selective Amplification of Bacterial mRNA

Original publication: Eriksson et al. (2003) Molecular Microbiology 47: 103–118“At each time point, infected macrophages were lysed on ice for 30 min in 0.1% SDS, 1% acidic phenol, 19% ethanol in water. The phenol-ethanol mixture acted to stabilize all bacterial RNA”

“The Chakraborty lab“Medical Microbiology, University Giessen, Germany

Selective lysis for highly enriched bacterial RNA - Intracellular Listeria and Salmonella

AFirst cDNA synthesisPreferential priming near the 3’-end of any nucleic acid sequence

Second cDNA synthesisPreferential priming near the 3’-end

A

BoxBox

BoxBox

BoxBox

BoxBox2

Third DNA synthesisBox-T7-promotor primer

BoxBox2 T7

Box

RNA removal

1 ng total RNA 2 amplificationrounds

> 50 µg amplified RNA

ExpressArt Selective Amplification of Bacterial mRNA

Agilent Bioanalyzer electropherograms of 2-rounds amplified E. coli mRNA

Hybridisation results Affymetrix E. coli Genome 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)>4,000

Suppression of rRNA amplification: less than 2% rRNAs in amplified RNAs

Differential gene expression in E. coli: Heat shockRNAs from E.coli grown at 37°C versus 50°CHybridisation with THE MWG E.coli K12 Array

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genes induced during heat shock

genes repressed during heat shock

Without amplification

Input 50 µg E.coli RNA Labelled cDNA with random primingHybridisation: 3µg labelled cDNA withHigh stringency

ExpressArt mRNA Amplification

Input 100 ng E.coli RNA

Hybridisation: 10µg labelled aRNA with standard conditions

Rosemarie Walter & James Eliason

Asterand plc

FFPE samples

John R. Arrand

Institute for CancerStudies

University of Birmingham,UK

Large and Small Samples

Degraded RNA Samples

ALL-EXON-ARRAYS

Ludger Klein-Hitpass

Institute forCell Biology(Tumor Research)Array Facility, Essen, Germany

Core Facility / FACS-Sorted Stem Cells

Craig P. Hunter & Ryan Baugh*Dept. of Molecular andCellular BiologyHarvard University, Cambridge, USAPicogram RNA Samples* now: lrb@caltech.edu

Bodo Brunneraventis-sanofi AG, Frankfurt, GermanyLarge and small samples

Chris Shepherd & David HudsonThe Cancer Research Institute, U.K.Degraded RNA Samples

Otto HagenbüchleISREC,Epalinges, SwitzerlandLarge and Small Samples

Edda Stoinski, MMI Zürich, SwitzerlandWolfgang Kemmner, MDC Berlin, GermanyLaser Microdissection / RNA isolation / RNA amplification / Microarrays

Edda Stoinski, MMI Zürich, SwitzerlandWolfgang Kemmner, MDC Berlin, GermanyLaser Microdissection / RNA isolation / RNA amplification / Microarrays

Renate Burgemeister & Ulrich SauerPALM, Bernried, GermanyLaser Microdissection / RNA isolation / RNA amplification / Microarrays

Renate Burgemeister & Ulrich SauerPALM, Bernried, GermanyLaser Microdissection / RNA isolation / RNA amplification / Microarrays

Estelle Marrer & Isabelle Keller

Veska Uzunova & Luther Sampson

NovartisPharma AG

FFPE samples & MMI Laser Microdissection

David Wong

Institute for Dentistry University of California, UCLA

ALL-EXON-ARRAYS: Degraded RNAs in Saliva

krupp@amp-tec.com www.amp-tec.com