a novel approach to selectively amplify mrna in challenging samples “expressart” dr. guido krupp...
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![Page 1: A novel approach to selectively amplify mRNA in challenging samples “ExpressArt” Dr. Guido Krupp The University of Vermont Burlington August 22, 2008](https://reader035.vdocument.in/reader035/viewer/2022070407/56649e395503460f94b2a4a4/html5/thumbnails/1.jpg)
A novel approach to selectively amplify mRNA in challenging samples
“ExpressArt”
Dr. Guido KruppAmpTec
Amplification Technologies
AmpTecAmplification Technologies
The University of VermontBurlingtonAugust 22, 2008
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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
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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?
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mRNA amplification technologiesProblems with very small samples
AmpTec ExpressArt® Technology*with two and three amplification rounds
* Courtesy: Baugh & Hunter, Harvard University
Mark
er
negat
ive co
ntrol:
no prim
er p
roducts
2 rou
nds
3 rou
nds
kb
3.0
0.5
0.2
Mark
er
negat
ive co
ntrol:
no prim
er p
roducts
2 rou
nds
3 rou
nds
kb
3.0
0.5
0.2
“Eberwine Method” with one and two amplification rounds
M
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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
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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
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Eberwine Scheme II: Transcription for first mRNA amplification round
T7
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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
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Short antisense RNAspreferrentially representing the 3‘-proximal section of mRNAs
T7
Eberwine Scheme IV: Transcription for second mRNA amplification round
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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
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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
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ExpressArt: Amplification of a model transcript
Input RNA800 nt mRNA
Amplified RNAExpressArt TRinucleotide primer
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ExpressArt: Amplification of a model transcript
Amplified RNAExpressArt TRinucleotide primer
Amplified RNArandom primer
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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
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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
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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
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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
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Mean foldchange ExpressArt 20ng 2x IVT
0.001
0.01
0.1
1
10
100
1000
0.001 0.01 0.1 1 10 100 1000
r = 0.983
Global foldchange analysis kidney vs. liver (4)
Mea
n f
old
chan
ge
Exp
ress
Art
2 n
g 3
x IV
T
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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
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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
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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
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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.”
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Heat your samples: 2 min @ 70°C
Watch out for your carrier!
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18
S
28
S
Flu
ore
sce
nce
Time (seconds)
-1
0
1
2
3
4
5
6
7
8
9
10
11
19 24 29 34 39 44 49 54 59 64 69
RNA without heating
18
S
28
S
Flu
ore
sce
nce
Time (seconds)
0
5
10
15
20
25
30
19 24 29 34 39 44 49 54 59 64 69
RNA with heating step
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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
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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
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Dr. Rosemarie Walter Research Lab at Asterand plc, Detroit, USA
RNA profiles: blue without red with NucleoGuard
Second “Helper“ for RNA recovery: NucleoGuard
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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
1
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.
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PAX5
18428 18403 18376 18425 184310
1
2
3 total-RNAaRNA
Fo
ld o
f P
ax5
exp
ress
ion
rela
ted
to
cas
e 18
428
ID3
18428 18403 18376 18425 184310
25
50
75 total RNAaRNA
Fo
ld I
D3
exp
ress
ion
rela
ted
to
cas
e 18
428
AICDA
18428 18403 18376 18425 184310
2
4
6
8
total RNAaRNA
50100150200250300
Fo
ld A
ICD
A e
xpre
ssio
nre
late
d t
o c
ase
1842
8
STAT6
18428 18403 18376 18425 184310
1
2
3 total RNA: no CT(STAT6) measuredaRNA
Fo
ld S
TA
T6
exp
ress
ion
rela
ted
to
cas
e 18
428
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
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SECOND → TRinucleotide priming for reverse transcription
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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
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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’
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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
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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
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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
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TRinucleotide mRNA AmplificationIntact versus Severely Degraded RNAr = 0.993
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TRinucleotide mRNA AmplificationIntact versus Severely Degraded RNAr = 0.993
Standard mRNA amplification with Oligo(dT)
r = 0.793
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Compact Structure of ribosomal RNAs
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ExpressArt® - The future of mRNA amplification
&High-Quality Microarrays
RNA from FFPE Samples
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“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
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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
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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“
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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
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Signal Intensities for Exon Probes over the Complete Length of GAP-DH mRNA
0
2
4
6
8
10
12
14
16
1 2 3 4 5 6 7 8 9
Exon
Sig
nal Affy Standard
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.
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Selective Amplification of Bacterial mRNA
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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
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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
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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
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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
aver
aged
rat
ios
of
two
dye
sw
ap s
lides
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
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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: [email protected]
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
[email protected] www.amp-tec.com