A Complete System for Formalin-Fixed SamplesThe Paradise® PLUS Reagent System provides all of the components necessary to prepare RNA from FFPE tissue samples for gene expression analysis. To help ensure successful microarray results, the system includes reagents optimized for exceptional recovery of RNA, the most sensitive RNA amplification available, and highly-efficient RNA labeling.

The Paradise® PLUS Reagent System has been validated as a key component of Life Technologies’ Systems for Microgenomics®. This reagent system has also been validated with all Applied Biosystems® Arcturus® laser capture microdissection (LCM) systems, which incorporate highly advanced technolo-gies for automated isolation of pure cell populations.

Benefits:

• Performgeneexpressionexperiments

usingarchivedtissueblocks

• Usemicroarraysorreal-timePCR

withsmallsamplesofRNAfromFFPE

material

• Reducecostandsavetimeby

prequalifyingsampleswiththequality

assessmentkit

• Usesamplespreparedfromavarietyof

fixationprotocols

The Applied Biosystems® Arcturus® Paradise® PLUS Reagent System from Life Technologies enables unprecedented gene expression analysis of formalin-fixed, paraffin-embedded (FFPE) samples. Using the Paradise® PLUS Reagent System, researchers can now measure whole gene expression profiles in these previ-ously inaccessible samples, using either microarray or quantitative real-time PCR (qPCR) analysis. The Applied Biosystems® Arcturus® Paradise® PLUS Reagent System includes reagents for sample preparation, tissue staining, RNA extraction, isolation, RNA amplification,and microarray labeling (Figure 1).

AppliedBiosystems®Arcturus®Paradise®PLUSReagentSystemFFPETissuestaining,extraction,isolation,amplification,andmicroarraylabelinginoneconvenientkit

PRODUCTBULLETINAppliedBiosystems®Arcturus®Paradise®PLUSReagentSystem

Figure 1. The Applied Biosystems® Arcturus® Paradise® PLUS Reagent System.

Figure 2. Gene Expression Profiling in Five Easy Steps.

2. Identify Cells of Interest

1. Prepare Samples

(1) Cut a 7 micron section from a paraffin block. De-paraffinize,

stain, and dehydrate tissue sections while keeping

RNA intact.

3. Extract and Isolate RNA

4. Amplify RNA

(2) Isolate 5,000 to 10,000 pure cells from FFPE tissue samples using either Applied Biosystems®

Arcturus® LCM systems or use full tissue scrapes from a slide.

(3) Recover high-quality total RNA using optimized

extraction andisolation reagents.

(4) Amplify as little as 5 ng of fixedRNA using methods that overcome challenges of crosslinked templates.

5. Profile Gene Expression

(5) Generate gene expressiondata using various compatible

microarray platforms.

Figure 3. Robust Data from FFPE Tissue Samples. (A) Paradise® PLUS Reagent System reproducibility on probe arrays. (B) Paradise® PLUS Reagent System reproduc-ibility on cDNA arrays. (C) Paradise® PLUS Reagent System reproducibility on oligonucleotide arrays.

Paradise® PLUS Reagent System reproducibility on GeneChip® Human Genome X3Parray (Affymetrix). Amplification was

performed on 5 ng of RNA from FFPE breast cancer samples.

Biotin-labeled samples were hybridized to duplicate GeneChip® microarrays,

resulting in intensity correlation of R = 0.983.

Paradise® PLUS Reagent System reproducibility on cDNA arrays. Two-round amplifications

were performed on independent laser captured FFPE tissue cells (~5,000 cells each). Amplified aRNA was labeled during reverse transcriptionand hybridized to two cDNA arrays, resulting

in correlation of gene expression ratios, R = 0.993 (log2 ratios).

Paradise® PLUS Reagent System reproducibility on oligonucleotide microarrays (Agilent Technologies). Two-round amplifications were performed on 5 ng

RNA obtained from independent laser-capturedFFPE human breast cells. Labeled aRNA washybridized to custom-designed microarrays,

resulting in correlation of gene expression ratios,R = 0.986 (log2 ratios).

Sample 1

cDNA Arrays

Sam

ple

2

R=0.993

–2.0 0.0 2.0

0.0

–2.0

2.0

Probe Arrays

Sample 1

Sam

ple

2

2 4 6 8 10 12

2

4

6

8

10

12R=0.983

Oligo Arrays

Sam

ple

2

R=0.986

B. C. A.

Sample 1

–2.0 0.0 2.0

0.0

–2.0

2.0

Complete Workflow for Gene ExpressionThe Paradise® PLUS Reagent System con-tains all the materials needed for a complete gene expression experiment (Figure 2). The kit includes components for:

• Staining the tissue to identify the cells of interest

• Extraction and isolation of total RNA from the cells of interest

• Amplification of isolated RNA to generate more material for gene expression assays

• Labeling for microarray experiments (optional)

Integrates Seamlessly Into Current Workflow The Paradise® PLUS Reagent System is compatible with all major microarray platforms. The RNA obtained using the Paradise® PLUS Reagent System can be used in the same fashion as RNA generated from frozen tissue sources. Robust and reproducible results are achieved using tools and techniques commonly employed in today’s research laboratories (Figure 3).

Ensure Downstream SuccessNot all FFPE samples contain high-quality RNA. Using the Paradise® PLUS QC Kit, FFPE tissue samples can be qualified before performing amplification and

microarray experiments. The QC kit’s simple qPCR assay ensures that the FFPE block contains usable RNA prior to investing the time and expense of LCM and RNA amplification (Tables 1 and 2). Researchers can then proceed in their study with confidence using these prequalified tissue samples (Figure 4).

Superior Applications and Technical SupportLife Technologies employs a team of highly qualified and expertly trained application scientists and technical support specialists. Our dedicated staff offers personalized training and application support, enabling researchers to thrive in any research area.

Table 1. A Comparison of RNA Recovery Yield Using Various FFPE Extraction Kits. Breast cancer tissue scrapes were run in triplicate using serial sections, each 1.0 cm x 1.1 cm. The Paradise® PLUS Reagent System gave the highest and most consistent yield compared to alternative FFPE RNA extraction kits. Data provided by M. Davey and R. Oullette, Atlantic Cancer Research Institute, Moncton, NB Canada.

RNA Yield Comparison

Company A Company B Company C Paradise® PLUS

Replicate Yield (ng) Yield (ng) Yield (ng) Yield (ng)

Day 1-1 1,173 756 155 8,235

Day 1-2 977 658 378 9,319

Day 2-1 2,342 351 384 9,185

Day 2-1 2,165 476 376 6,836

OverallMean

1,664 560 323 8,394

SD 688.9 181.4 112.2 1145.1

%CV 41.4 32.4 34.7 13.6

Figure 4. Comparative Microarray Data. Log2 scatter plots generated using raw fluorescence intensity data from Affymetrix GeneChip® arrays, with Pearson correlation coefficients calculated for (A) frozen LCM replicates, r = 0.970; (B) FFPE LCM replicates, r = 0.961; (C) FFPE scrape compared to LCM, r = 0.932; and (D) frozen LCM compared to FFPE LCM, r = 0.937. Genes that fall on the green line have equal intensities on both arrays, and genes falling outside the red lines have a 2-fold or greater difference in intensity between the two chips. The technical reproducibility of FFPE samples is almost identical to that of frozen samples. The decrease in correlation between the FFPE scrape and LCM reflects the gene expression differences between mixed cells and pure cells, highlighting the benefit LCM provides over tissue scrapes to isolate more pure samples. A similar decrease in correlation is seen between frozen and FFPE samples (D) compared to replicate frozen (A) or FFPE (B) samples. However, the correlation is sufficiently high (r = 0.937) to enable reliable gene expression profiling of FFPE samples to represent expression levels in the tissue’s native state.

Table 2. A Comparative qPCR Study of Frozen Tissue vs. FFPE Tissue Using Various FFPE RNA Extraction Kits. Each sample was run in triplicate. Real-time PCR analysis was performed on the samples using the housekeeping gene GAPDH. The same amount of frozen and FFPE RNA was put into each reaction. The Paradise® PLUS Reagent System gave the lowest delta Ct difference values between the frozen mate-rial and the FFPE material (Ct FFPE – Ct frozen), indicating that RNA extracted from the Paradise® PLUS Reagent System most accurately represents the RNA extracted from pristine frozen material. Data provided by M. Davey and R. Oullette, Atlantic Cancer Research Institute, Moncton, NB Canada.

Frozen vs. FFPE Comparison

Company A Company B Company C Paradise®PLUS

ReplicateDelta Ct Frozen

vs. FFPE

Delta Ct Frozen

vs. FFPE

Delta Ct

Frozenvs. FFPE

Delta Ct Frozenvs. FFPE

Day 1-1 10.2 10.3 8.7 8.1

Day 1-2 10.2 10.8 12.3 7.4

Day 2-1 8.0 9.4 10.4 7.6

Day 2-1 8.5 10.6 12.5 7.8

OverallMean

9.21 10.27 10.95 7.73

SD 1.2 0.6 1.8 0.3

%CV 12.6 6.3 16.4 4.0

For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.

© 2010 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners. Cy is a registered trademark of GE Healthcare.

Printed in the USA. 07/2010 CO13966

ORDERING INFORMATION

Description Size Part Number

Paradise® PLUS Reagent System 12 two-round reactions KIT0312

Paradise® PLUS Reagent System with Biotin Labeling 12 two-round reactions KIT0312B

Paradise® PLUS Reagent System with Cy®3 Labeling 12 two-round reactions KIT0312C

Paradise® PLUS Reagent System with Cy®5 Labeling 12 two-round reactions KIT0312D

Paradise® PLUS Reagent System with Amino Allyl Labeling 12 two-round reactions KIT0314

Paradise® PLUS Reagent System for Quantitative Real-time PCR 12 two-round reactions KIT0310

Paradise® PLUS Reagent System for Quality Assessment of FFPE Block (QC Kit) 12 reactions KIT0313

Paradise® PLUS Whole Transcript RT Reagent SystemNote: Many other configurations are available customized for your research needs

12 reactions KIT0315

PEN Membrane Frame Slides 50 slides LCM0521

PEN Membrane Glass Slides 50 slides LCM0522

CapSure® Macro LCM Caps 48 caps LCM0211

CapSure® HS LCM Caps 32 caps LCM0214

Headquarters850 Lincoln Centre Drive | Foster City, CA 94404 USA Phone 650.638.5800 | Toll Free 800.327.3002 www.appliedbiosystems.com

International Sales For our office locations please call the division headquarters or refer to our Web site at www.appliedbiosystems.com/about/offices.cfm

For additional information on the Applied Biosystems® Arcturus® Paradise® PLUS Reagent System and to place your order, visit www.appliedbiosystems.com/arcturus.

Choose the Product Configuration forYour ApplicationThe Paradise® PLUS Reagent System is available with options customized for your research:

• Biotin labeling• Cy®3 labeling• Cy®5 labeling• Aminoallyl incorporation• Natural nucleotides• Real-time PCR

For Probe Arrays Requiring Biotin-LabeledMaterial:• Perform two rounds of amplification

generating amplified RNA (aRNA)• Use the Applied Biosystems® Arcturus®

Turbo Labeling™ Kit to label enough material with biotin to hybridize to your array*

• Remaining aRNA is unlabeled for further comparison or validation (real-time PCR or alternative array platforms)

For Fluorescent and Two-Color Arrays:• Perform 2 rounds of amplification

generating aRNA

• Use Applied Biosystems® Arcturus® Turbo Labeling™ Kit to label enough material with Cy®3 or Cy®5 dye to hybridize to your array*

• Remaining aRNA is unlabeled for further comparison or validation (real-time PCR or alternative array platforms)

For qRT-PCR Analysis:• Perform one round of amplification• Convert amplified RNA to cDNA• cDNA is ready for real-time PCR

* Other labeling options also available