paradise reagent system - harvard university · 5. remove section(s) from microtome and float them...

Version A 888.446.7911 650.962.3020 [email protected] www.arctur.com

ParadiseReagent System

Catalog # KIT0301, KIT0302

User Guide

Version AFor Laboratory Use

p/n 12872-00

Paradise Reagent System




User Guide, Version A


Preface

Section 1: Sample Preparation and StainingI. Introduction 1-4II. Components 1-5III. Preliminary Steps 1-6IV. Protocol 1-8V. Troubleshooting 1-11VI. Appendix 1-12

Section 2: RNA Extraction / IsolationI. Introduction 2-5II. Components 2-6III. Preliminary Steps 2-7IV. Protocol 2-8V. Troubleshooting 2-13

Section 3: RNA AmplificationI. Introduction 3-5II. Components 3-7III. Preliminary Notes 3-8IV. Protocol 3-10V. Appendices 3-27VI. Troubleshooting 3-36

Table of Contents

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Legal Notices

Copyright © 2003 Arcturus Bioscience, Inc. All Rights Reserved.

PixCell®, CapSure® and RiboAmp® are registered trademarks, and HistoGene,PicoPure, MiraCol, AutoPix and Paradise are trademarks of Arcturus Bioscience,Inc. Other trademarks used in this manual are the property of their respective owners.The PCR process is covered by patents owned by Hoffmann-La Roche Inc. and F.Hoffman-La Roche Ltd. Some uses of the Paradise Reagent System may requirelicenses from third parties. Purchase of the Paradise Reagent System does notinclude any right or license to use, develop or otherwise exploit the productcommercially. Any commercial use, development or exploitation of the ParadiseReagent System or development using the product without the express writtenauthorization of Arcturus Bioscience, Inc. is strictly prohibited.

Warranty

Arcturus Bioscience, Inc. warrants that the products described in this manual meet theperformance standards described in literature published by the company. If a productfails to meet these performance standards, Arcturus will replace the product or issuecredit for the full purchase price, including delivery charges. Arcturus provides noother warranties of any kind, expressed or implied. Arcturus’ warranty liability shallnot exceed the purchase price of the product and shall not extend to direct, indirect,consequential or incidental damages arising from the use, results of use, or improperuse of its products. The Paradise Reagent System is intended for laboratory use.

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Version A 888.446.7911 650.962.3020 [email protected] www.arctur.com i i ii i ii i ii i ii i i

The Paradise Reagent System provides an integrated system enabling gene expression studies using Formalin-Fixed Paraffin-Embeddedtissue (FFPE). The components provided with the system include:

• Sample preparation and staining reagents• RNA extraction and isolation reagents• RNA amplification reagents

Each section of this user guide describes the steps involved using each of these. The reagents included with the Paradise System are intendedto be used together as part of the integrated system. Alternate reagents should not be substituted as the reagents and process provided havebeen developed for optimal performance. All reagents included with the system should be used prior to the expiration date clearly markedon the package label.

To get the most out of the Paradise Reagent System please examine the components and read through each section of the user guidecarefully.

Related DocumentsWhen using the Paradise Reagent System User Guide, the following user guides may be helpful references:

• AutoPix LCM System User Guide• PixCell IIe LCM System User Guide• CapSure HS User Guide

Quality ControlArcturus performs functional testing on all components of the Paradise Reagent System. The information sheet provided with the systemhighlights the tests performed.

Technical SupportYou may access services and support in the following ways:

• By online connection at www.arctur.com,• By phone at 888-446-7911 (US toll-free) or 650-962-3020• By email to [email protected]

Comments and SuggestionsArcturus welcomes your comments and suggestions for improving our documents. Send your comments to [email protected].

Preface

Arcturus400 Logue AvenueMountain View, CAUSA 94043www.arctur.com

Arcturus SARLFrance[33] (1) 300-75 979 tel[33] (1) 300-75 651 [email protected]

Arcturus GmbHGermany[49] (0) 61 05-40 88 0 tel[49] (0) 61 05-40 88 40 [email protected]

Arcturus Ltd.United Kingdom[44] (158) 246-9010 tel[44] (158) 246-7988 [email protected]

888.446.7911650.962.3020 tel650.962.3039 [email protected]


Section 1:

Sample Preparation and Staining

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Version A 888.446.7911 650.962.3020 [email protected] www.arctur.com1-21-21-21-21-2


I. IntroductionA. Background 1-4B. Storage and Stability 1-4C. Material Safety and Data Sheets 1-4

II. ComponentsA. Reagents and Supplies 1-5

III. Preliminary StepsA. Material and Protocol Review 1-6B. Recommendations for RNase-free Technique 1-6C. Additional Equipment and Materials Required 1-6

IV. ProtocolA. Slide Preparation 1-8B. Deparaffinization, Staining and Dehydration 1-9

V. TroubleshootingA. Targeted Cells do not Lift from the Slide During LCM 1-11B. RNA Cannot be Recovered from the Sample 1-11

VI. AppendixA. Related Protocols 1-12


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I. Introduction

A. Background

A principal application of Laser Capture Microdissection (LCM) is theanalysis of gene expression patterns in cells captured from specimens.Obtaining accurate results from gene expression analysis experiments,including microarray hybridization and quantitative PCR, depends oncareful preservation of intact RNA molecules in captured cells.

The Paradise Reagent System Staining components are part of a series ofLCM-certified LCM analysis products for preparing and staining tissueswhile preserving intact nucleic acid and protein species from captured cellpopulations. The staining components work with the additional modulesprovided in this reagent system. Paradise extraction and isolation reagentsand RNA amplification reagents provide a complete solution for studyingRNA from cells isolated by LCM. The reagents and protocol have beenoptimized for use with Formalin-Fixed Paraffin-Embedded (FFPE)samples.

Arcturus maintains an ongoing research program to test and validate theParadise Reagent System. Call Arcturus Technical Support at (888) 446-7911 or (650) 962-3020, or send an email inquiry [email protected] for an up-to-date list.

B. Storage and Stability

Inspect all kit components upon receipt. Ethanol and xylene are flammableand should be unpacked and stored at room temperature in a fireproofstorage cabinet or fume hood with adequate ventilation. Cap bottles tightlybetween uses. Store remaining kit supplies at room temperature in a clean,dust-free environment.

C. Material Safety and Data Sheet

Material Safety and Data Sheets (MSDS) for kit chemical components areavailable from the the Arcturus web site at www.arctur.com. They may alsobe acquired by calling Arcturus Technical Services at (888) 446-7911 or(650) 962-3020, or by sending e-mail to [email protected].

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II. Components

A. Reagents and Supplies

The Paradise Reagent System Staining components include:

Description Units

q 100% Ethanol 1 Lq 95% Ethanol 1 Lq 75% Ethanol 1 Lq Xylene 1 Lq Distilled Water Nuclease Free 500 mlq Staining Solution 6 mlq Staining Jars 10


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III. Preliminary Steps

A. Material and Protocol Review

To get the most from your staining reagents, take a few moments to examinethe components of the kit and read through the information in the followingsections.

B. Recomendations for RNase-free Technique

RNase contamination will cause experimental failure. Minimize RNasecontamination by adhering to the following recommendations throughoutyour experiment:

· Wear disposable gloves and change them frequently.

· Use RNase-free solutions, glassware and plasticware.

· Do not re-purify Paradise Reagent System Section Staining Kitcomponents. They are certified Nuclease Free.

· Wash scalpels, tweezers and forceps with detergent and bake at 210°Cfor four hours before use.

· Use RNase AWAY® (Life Technologies) according to the manufacturer’sinstructions on the horizontal staining rack and any other surfaces thatmay come in contact with the sample.

· Use Kimwipe soaked in RNase Away to wipe down and clean theinterior of tissue flotation water bath.

C. Additional Equipment and Materials Required

Ensure that you have ready access to the following laboratory equipmentand materials before you begin. These items are not included with theParadise Reagent System:

1. Equipmentq Rotary Microtomeq Fume hoodq –70°C freezerq Tweezersq Cover glass forcepsq Microslide box – plastic (VWR Cat. # 48444-004)q Tissue Flotation Water Bathq Ovenq 20 – 200 µl pipettor

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2. Materialsq Disposable glovesq Detergent (Fisher Scientific, Cat. # 04-355q RNase AWAY (Life Technologies, Cat. # 10328-011)q 100% ethanolq Kimwipes® or similar lint-free towelsq Disposable microtome bladesq Microslidesq Pipette tips, nuclease free

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IV. Protocol

A. Slide Preparation

1. Prior to starting slide preparation, minimize RNase contamination ofthe equipment by cleaning as follows:

a. Rotary Microtome: Remove and discard old disposablemicrotome blade. Use a Kimwipe soaked with RNase Away towipe down the knife holder. Dry holder with a clean kimwipe.Install a new disposable microtome blade into holder.

b. Tissue Floatation Bath: Use a Kimwipe soaked with RNase Awayto wipe down and clean the interior of the water bath. Rinse theinterior with Milli-Q or RNase free water. Fill the water bathwith Milli-Q or RNase free water. Heat water to appropriatetemperature for the paraffin used in your laboratory, typically5–10oC below its melting point. Do not add any adhesives tothe water bath.

2. Set cutting thickness to 7 µm on the microtome.

3. Place paraffin block into specimen holder. Trim off any excess paraffinfrom the block face. Cut and discard the first five sections aftertrimming.

4. From the fresh surface, cut 7 µm sections from your specimen. If youare cutting more than one specimen, use a new disposable blade foreach one to avoid cross contamination.

5. Remove section(s) from microtome and float them onto heated waterbath. Allow section(s) to flatten. Minimize time in water bath to nolonger than 2 minutes. Mount each section on a room-temperatureslide.

6. Prop slide on end to allow water to drain away from section. Air-drythe slide for a minimum of 30 minutes at room temperature. Discardany slides that have wrinkles or folds in the section.

7. Proceed immediately to the Deparaffinization, Staining andDehydration segment of the protocol or store slides at –70oC in amicroslide box for up to two weeks.

8. After completion of the slide preparation process, remove any paraffindebris from the microtome. Clean surfaces with a kimwipe soakedwith RNase Away and dry all surfaces. Discard water from water bathand clean the interior with RNase Away and dry all surfaces.

Wear clean disposable gloves throughout the Slide Preparationprocedure. Use clean RNase-free instruments. Wear cleandisposable gloves throughout the Slide Preparation procedure.

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Depending on humidity in the environment, drying may takeup to 90 minutes. The section must be dry before proceeding.


B. Deparaffinization, Staining and Dehydration

1. Label 10 plastic slide jars as follows:a. Xyleneb. Xylenec. 100% ethanold. 95% ethanole. 75% ethanolf. Nuclease free waterg. 75% ethanolh. 95% ethanoli. 100% ethanolj. xylene

2. Using the LCM-certified solutions provided, fill the labeled plasticslide jars with 25 ml of the appropriate solution.

3. Remove up to four slides from the slide box or from the –70°Cfreezer, and place in a 50–60°C oven for 2 minutes.

4. Place the slides in plastic slide jar “a” containing xylene for 2 minutes.Invert jar gently.

5. Transfer the slides to plastic slide jar“b” containing xylene for 2minutes. Invert jar gently.

6. Transfer the slides to plastic slide jar “c” containing 100% ethanol for2 minutes. Invert jar gently.

7. Transfer the slides to plastic slide jar “d” containing 95% ethanol for1 minute.

8. Transfer the slides to plastic slide jar “e” containing 75% ethanol for 1minute.

9. Transfer the slides to plastic slide jar“f” containing nuclease free waterfor 30 seconds.

10. Using an RNase free pipette tip, apply 100 µl of the Paradise StainingSolution so that it covers the entire section. Stain for 45 seconds atroom temperature. Tap off excess stain before proceeding with thefollowing steps.

11. Transfer the slides to plastic slide jar “g” containing 75% ethanol for30 seconds.

12. Transfer the slides to plastic slide jar “h” containing 95% ethanol for30 seconds.

13. Transfer the slides to plastic slide jar “i” containing 100% ethanol for1 minute.

Carry out the Staining and Dehydration segment of the protocolin a hood. Wear clean disposable gloves.

Xylene jar “a” must be changed after processing up to a maximumof 4 slides.

75% Ethanol jar “e”must be changed after processing up to amaximum of 4 slides.

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14. Transfer the slides to plastic slide jar “j” containing xylene. Hold slidesin xylene until ready for microdissection. The minimum incubationin xylene should be 5 minutes, or up to a maximum of 2 hours.

15. Place the slides on a Kimwipe to dry in the hood for five to tenminutes prior to LCM. LCM should be performed within 2 hoursafter removal from xylene.

16. Discard all used staining and dehydration solutions according tostandard procedures.

Performing Laser Capture Microdissection (LCM)

Please consult the User Guide for the instrument you will use for detailedinstructions.

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V. Troubleshooting

A. Targeted cells do not lift from the slide duringLCM

1. The sample may contain residual water. Ensure that the ethanol solutionsare fresh. Ethanol is hygroscopic. Keep the ethanol bottles tightlycapped, and do not pour ethanol solutions until you are ready to usethem. If you suspect that the 100% ethanol solution has absorbedwater, discard and use a new bottle.

2. The sample may have dried in between protocol steps. Carry out theStaining and Dehydration segment of the protocol at a steady pace.

B. RNA cannot be recovered from the sample

1. The sample starting material may contain poor quality RNA.

2. RNA may become degraded during RNA isolation. Wear gloves; useRNase-free technique and RNase-free instruments and reagents.

3. RNA may not be fully extracted and isolated from cells on the LCMcap. Perform RNA extraction immediately after LCM to ensurecomplete extraction and optimum recovery of RNA.

4. Amount of starting material may be insufficient.

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VI. Appendix

A. Related Protocols

1. Cleaning the staining jars

a. The staining jars can be reused, but must be cleaned. Rinse jarswith 100% ethanol, followed by distilled water, then treat withRNase AWAY according to the manufacturer’s protocol. Rinsejars thoroughly with nuclease-free water and allow to drycompletely in the hood. Do not use reservoirs to store solutions.

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Section 2:

RNA Extraction / Isolation

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I. IntroductionA. Background 2-5B. Performance Specifications 2-5C. Quality Control 2-5D. Storage and Stability 2-5E. Material Safety and Data Sheets 2-5


III. Preliminary StepsA. Recommendations for RNase-free Technique 2-7B. Recommendations for Storing RNA 2-7C. Additional Equipment and Materials Required 2-7

IV. ProtocolA. Overview 2-8B. Protocol for Use with CapSure HS LCM Caps 2-9C. Tissue Scrape Protocol 2-11

V. TroubleshootingA. Isolated RNA is of Poor Quality 2-13B. RNA Yield is Low 2-13

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I. Introduction

A. Background

The Paradise Reagent System RNA Extraction/Isolation reagents enableresearchers to recover total cellular RNA from formalin fixed paraffinembedded samples. They are optimized for use with cells acquired usingLaser Capture Microdissection (LCM) on CapSure HS LCM Caps. Totalcellular RNA isolated using the Paradise Reagent System RNA Extraction/Isolation reagents produces RNA in a small volume of low ionic strengthbuffer, ready for use in linear amplification using the Paradise ReagentSystem RNA amplification reagents. The Paradise Reagent System RNAExtraction/Isolation Kit contains RNA extraction and purification reagentsand MiraCol Purification Columns.

B. Performance SpecificationsResearchers can complete total cellular RNA isolations from cells capturedon CapSure HS LCM Caps . RNA extraction from LCM caps requiresovernight incubation (16 hours); the RNA purification process takes lessthan 35 minutes.

Reagents are provided for isolating total RNA from a 0.5 cm x 0.5 cmtissue scrape.

C. Quality ControlArcturus performs functional testing on the Paradise Reagent System RNAExtraction/Isolation using all components. MiraCol Purification Columnsare tested by lot to confirm the absence of nucleic acids and nucleaseactivity. Column nucleic acid binding and recovery performance mustmeet quality standards.

D. Storage and StabilityStore the Paradise Reagent System RNA Extraction/Isolation componentsat room temperature. Store the DNase I solution, DNase Buffer andReconstitution Buffer at –70ºC until use. Once the reagents are used,storage at –20ºC is recommended. Properly stored reagents are stableuntil the expiration date indicated on the package.

E. Material Safety and Data SheetsMaterial Safety and Data Sheets (MSDS) for kit chemical components areavailable from Arcturus Technical Services. Call 888.446.7911 or650.962.3020, send e-mail to [email protected], or download thefile from www.arctur.com.


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II. Components

A. Reagents and SuppliesThe Paradise Reagent System RNA Extraction/Isolation componentsinclude the following items:

Item Vial Name

q Conditioning Buffer CBq Ethanol Solution EtOHq Wash Buffer 1 W1q Wash Buffer 2 W2q Elution Buffer EBq Pro K Mixq Reconstitution bufferq Binding Buffer BBq DNase Mix DNaseq DNase Buffer DNBq MiraCol RNA Purification

Columns with collection tubesq Microcentrifuge tubes

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III. Preliminary Steps

A. Recomendations for RNase-free Technique


• Always handle RNA in a manner that avoids introduction of RNases.

• Wear disposable gloves and change them frequently to prevent theintroduction of RNases from skin surfaces.

• After putting on gloves, avoid touching surfaces that may introduceRNases onto glove surfaces.

• Do not use reagents not supplied with the Paradise Reagent System.Substitution of reagents or Kit components may adversely affect yieldsor introduce RNases.

• Use only new plasticware that is certified nucleic acid-free.

• Use only new, sterile, RNase-free pipette tips and microcentrifugetubes.

• Clean work surfaces with commercially available RNasedecontamination solutions prior to performing reactions.

B. Recommendations for Storing RNA

Begin the RNA Extraction/Isolation protocol immediately followingacquisition of cells by LCM. Following protocol completion, use isolatedRNA immediately for amplification, or store at –70°C or below for up to6 months.

C. Additional Equipment and Materials Required

Ensure that you have ready access to the following laboratory equipmentand materials before you begin. These items are not included with the RNAExtraction/Isolation components:

1. Equipmentq Microcentrifuge (Eppendorf 5415D or similar)q 2–20 µL pipettorq 20–200 µL pipettorq Incubation oven (50°C)

2. Materialsq Nuclease-free pipette tips


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IV. Protocol

A. Overview

Separate protocols are provided for extraction/isolation of RNA frommicrodissected samples (Section B) or from 0.5 cm x 0.5 cm tissue scrapes(Section C).

The flow chart illustrates the Paradise Reagent System RNA Extraction/Isolation procedure: (a) Extract RNA from a CapSure HS LCM Cap. (b)Mix and load cell extract onto a preconditioned purification column. (c)Spin the extract through the column to capture RNA on the purificationcolumn membrane. Wash, DNase treat, and wash again. (d) Wash thecolumn twice with wash buffer, and (e) elute the RNA in low ionic strengthbuffer. The entire isolation process, including incubations, can be completedin less than an hour, and the isolated total cellular RNA is ready for use indownstream applications. The Paradise Reagent System RNA Extraction/Isolation reagents are capable of isolating small amounts of RNA. It isimportant not to introduce nucleic acid contamination.

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B. Protocol for Use with CapSure HS LCM Caps

1. RNA Extraction

a. Dispense Pro K Mix and incubate as follows:

i. Capture cells and assemble the CapSure HS Cap with theExtracSure Extraction Device. Refer to the CapSure HS CapsUser Guide for complete instructions.

ii. Thaw the Reconstitution Buffer, mix thoroughly with all solidsdissolved, and maintain at 4°C.

iii. Add 75 µl of Reconstitution Buffer to vial of dried Pro K Mix.Dissolve completely. One vial of Pro K Mix is adequate for 7extractions.

iv. Place the CapSure–ExtracSure assembly in a CapSure HSAlignment Tray and pipette 10 µL Pro K Mix solution into thebuffer well. Place pipette tip down to the film surface to avoidtrapping a bubble.

v. Place a new 0.5 mL microcentrifuge tube onto the CapSure–ExtracSure assembly. Cover with Incubation Block.

vi. Incubate assembly for 16 hours at 50°C.

b. Centrifuge the microcentrifuge tube with the CapSure–ExtracSureassembly at 800 x g for two minutes to collect cell extract into themicrocentrifuge tube.

c. After centrifugation, the microcentrifuge tube contains the cell extractrequired to complete the protocol. Remove the microcentrifuge tubefrom the CapSure–ExtracSure assembly and save the microcentrifugetube with the cell extract in it.

d. Proceed with RNA isolation protocol or freeze cell extract at –70°C.

Pipettor Tip

ExtracSureSampleExtractionDevice

Heating Block

CapSure–ExtracSureAssembly withMicrocentrifugeTube

Alignment Tray

It is okay to stop at this point in the protocol.If multiple LCM captures are performed, it is recommendedthat each cap be incubated immediately after collection in ProK Mix. Caps may be incubated up to 24 hours.


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2. RNA Isolation

a. Pre-condition the MiraCol Purification Column as follows:

i. Pipette 200 µl Conditioning Buffer (CB) onto the purificationcolumn filter membrane.

ii. Incubate the purification column with Conditioning Buffer for5 minutes at room temperature.

iii. Centrifuge the purification column in the provided collectiontube at 16,000 x g for one minute.

b. Pipette 35 µl of Paradise Reagent System binding buffer (BB) intothe cell extract from Part 1 (RNA Extraction). Mix well by pipettingup and down. DO NOT CENTRIFUGE. Pipette 65 µl of EthanolSolution (EtOH) into tube and mix well.

c. Pipette the cell extract mixture into the preconditioned purificationcolumn. The cell extract mixture will have a combined volume ofapproximately 110 µl.

d. To bind RNA, centrifuge for 2 minutes at 100 x g, immediatelyfollowed by a centrifugation at 16,000 x g for 1 minute.

e. Pipette 100 µl Wash Buffer 1 (W1) into column and centrifuge for1 minute at 8000 x g.

f. Mix 2 µl DNase Mix (DNase) with 18 µl of DNase buffer (DNB).Add 20 µl mixture to the column and incubate at room temperaturefor 20 minutes.

g. Pipette 40 µL Wash Buffer 1 (W1) into the purification column andcentrifuge for one minute at 8000 x g.

h. Pipette 100 µL Wash Buffer 2 (W2) into the purification columnand centrifuge for one minute at 8000 x g.

i. Pipette another 100 µL Wash Buffer 2 (W2) into the purificationcolumn and centrifuge for two minutes at 16,000 x g. Check thepurification column for any residual wash buffer. If wash bufferremains, recentrifuge at 16,000 x g for one minute.

j. Transfer the purification column to a new 0.5 mL microcentrifugetube provided.

k. Pipette12 µl Elution Buffer (EB) directly onto the membrane of thepurification column (Gently touch the tip of the pipette to the surfaceof the membrane while dispensing the elution buffer to ensuremaximum absorption of EB into the membrane).

l. Incubate the column for one minute at room temperature.

m. Place each column tube assembly into the 2 ml support tube in therotor with the 0.5 ml tube cap trailing the tube.

To avoid potential breakage of the microcentrifuge tube capduring centrifugation, insert the purification column/ 0.5 mLtube assembly into a lidless 2.0 mL tube. Insert this assemblyinto adjacent rotor holes as illustrated. Rest the tube cap againstthe tube immediately clockwise to it. Place an empty, lidless 2.0mL tube into the rotor hole adjacent in the clockwise direction tothe last assembly.

Flowthrough waste following centrifugation is usually presentas only a small volume, and therefore it is not necessary todiscard the flowthrough waste after every centrifugation step.Make sure that the accumulated flowthrough waste does notmake contact with the purification column. Flowthrough wasteshould be discarded when the waste fluid level approaches thesurface of the purification column.

Remove all traces of wash buffer prior to transferring purificationcolumn to the new microcentrifuge tube. To remove wash buffer,discard flowthrough waste and recentrifuge the column for oneminute at 16,000 x g.

Prior to use, mix Binding Buffer (BB) thoroughly. BindingBuffer (BB) may form precipitate upon storage. Dissolve precipitateprior to use by mixing thoroughly. If necessary, warm the BB vialto redissolve Binding Buffer prior to use.

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n. Centrifuge the column for one minute at 1,000 x g to distribute EBin the column, and then spin for one minute at 16,000 x g to eluteRNA. The entire sample may be used immediately or stored at–70°C or below.

C. Tissue Scrape Protocol

Follow Section Staining Protocol to prepare the tissue scrape sample.

1. Scrape and RNA Extraction

a. Prepare 1 vial of Pro K Mix by adding 75 µl reconstitution buffer toa vial of dried Pro K Mix. Dissolve completely.

b. Pipette 25 µl Pro K solution into a DNase, RNase free 0.5 mlmicrocentrifuge tube.

c. Using a clean, sterile scalpel blade, take the dried slide and scrape offan area not exceeding 0.5 cm x 0.5 cm from the section and place thescrape into the microcentrifuge tube containing the 25 µl ProteinaseK solution.

d. Vortex slightly. Visually inspect to ensure that the tissue scrape is inthe Pro K solution and not stuck to the side of the microcentrifugetube.

e. Incubate tube in an oven set to 50oC for 16 hours.

f. Proceed to RNA isolation or store at –70oC or below.

2. RNA Isolation

a. Pre-condition the MiraCol Purification Column as follows:

i. Pipette 200 µl Conditioning Buffer (CB) onto the purificationcolumn filter membrane.

ii. Incubate the purification column with Conditioning Buffer for5 minutes at room temperature.

iii. Centrifuge the purification column in the provided collectiontube at 16,000 x g for one minute.

b. Pipette 87.5 µl of Paradise Reagent System binding buffer (BB) intothe cell extract from Part 1 (RNA Extraction). Mix well by pipettingup and down. DO NOT CENTRIFUGE. Pipette 162.5 µl ofEthanol Solution (EtOH) into tube and mix well.

c. Centrifuge the mixture at 16000 x g for 1 minute to pellet the debris.

d. Load 200 µl of supernatant from the mixture, taking care not todisrupt the pellet, into the preconditioned purification column. Tobind RNA, centrifuge at 100 x g for 2 minutes, then at 16,000 x g for1 minute.

One vial of proteinase K is adequate for 3 tissue scrapesamples.

Use a new scalpel blade for each sample to avoid cross-contamination.

Discard flowthrough waste when the waste fluid levelapproaches the bottom surface of the purification column.


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e. Repeat step d until all of sample mixture has been loaded andcentrifuged.

f. Pipette 100 µl Wash Buffer 1 (W1) into column and centrifuge at16,000 x g for 1 minute.

g. Mix 2 µl DNase Mix (DNase) with 18 µl of DNase buffer (DNB).Add 20 µl mixture to the column and incubate at room temperaturefor 20 minutes.

h. Pipette 40 µL Wash Buffer 1 (W1) into the purification column andcentrifuge for one minute at 8000 x g.

i. Pipette 100 µl Wash Buffer 2 (W2) into column and centrifuge at16,000 x g for 1 minute.

j. Pipette 100 µl Wash Buffer 2 (W2) into column and centrifuge at16,000 x g for 2 minutes.

k. Transfer column to a 0.5ml microcentrifuge tube provided in theKit.

l. Pipette 70 µl of Elution Buffer (EB) direction onto the membrane ofthe purification column.

m. Incubate for 1 minute at room temperature.

n. Centrifuge at 1,000 x g for 1 minute and then at 16,000 x g for 1minute. The sample maybe used immediately or stored at –70oC orbelow.

Gently touch the tip of the pipette to the surface of themembrane while dispensing the elution buffer to ensuremaximum absorption of EB to the membrane.

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V. Troubleshooting

A. Isolated RNA is of Poor Quality

1. Verify quality of source tissue of LCM cells. The greatest factor affectingthe quality of isolated RNA is the integrity of the RNA in the originaltissue sample. RNA degradation due to RNase activity occurs rapidly,especially upon tissue removal such as through biopsy and needleaspiration. For suggestions on verifying quality, please call ArcturusTechnical Support.

2. Use the Paradise Reagent System Staining components to prepareslides for LCM. Specialized staining protocols and reagents are requiredfor optimal RNA preservation in LCM samples. Arcturus has developedand validated the Paradise Reagent System Staining components forpreparing and staining tissues for LCM while maintaining RNAintegrity.

3. Perform LCM immediately after preparing LCM slides. LCM sampleslides are dehydrated in the final step of preparation so RNase activityis minimized. However, the risk of moisture and RNases entering thesample following preparation increases with the amount of timebetween slide preparation and RNA isolation.

4. Use FFPE sections that are within 2 weeks of cutting. Extendedstorage of sections after being cut from blocks may result in RNAdegradation.

B. RNA Yield is Low

1. RNA integrity has been compromised. Verify quality of initial tissuesample or LCM slide (see A.1). Poor quality RNA may not bindeffectively to the purification column membrane, decreasing overallRNA yield.

2. Buffer concentrations in extraction mixtures are incorrect due toinadequate mixing with Ethanol Solution. Ensure all buffers arecompletely mixed and all solids are dissolved prior to use.

3. Elution Buffer (EB) concentration is incorrect due to contaminationwith Wash Buffer 2 (W2). Ensure that all Wash Buffer 2 has beenremoved by centrifugation before proceeding to add Elution Buffer.Residual Wash Buffer 2 (W2) on the purification column filtermembrane will alter the concentration of Elution Buffer (EB),resulting in poor RNA elution. If any Wash Buffer 2 (W2) remains inor on the purification column, recentrifuge it to remove the residualbuffer before proceeding to elution.

4. Extraction step incubation was too short. Incubate the LCM samplein Pro K solution for a full 16 hours at 50°C. Complete cell extractionfrom fixed, dehydrated samples requires this validated incubationcondition. Samples may be incubated for up to 24 hours.


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Section 3:

RNA Amplification

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I. IntroductionA. Background 3-5B. Performance Specifications 3-5C. Quality Control 3-5D. Storage and Stability 3-6E. Material Safety and Data Sheets 3-6


III. Preliminary NotesA. Recommendations for RNase-free Technique 3-8B. Amplified aRNA Contamination 3-8C. RNA Preparation 3-8D. RNA Storage 3-9E. Additional Equipment and Materials Required 3-9

IV. ProtocolA. Overview 3-10B. Thermal Cycler Programming 3-11C. Time Requirements 3-11D. Detailed Protocol 3-13

V. AppendicesA. Applications of aRNA 3-27B. aRNA Yield and Purity Determination 3-34C. Assessment of RNA Sample Quality by Quantitative Real-Time PCR 3-34D. Assessment of aRNA Quality Using the Agilent Lab-on-a-Chip System 3-35E. Analysis of aRNA by Agarose Gel Electrophoresis 3-36

VI. TroubleshootingA. Amplification Yield is Poor 3-37B. Low Molecular Weight Product Appears on Gel 3-38

RNA Amplification

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I. Introduction

A. Background

The Paradise Reagent System RNA Amplification components provideunparalleled sensitivity in linear amplification of formalin fixed paraffinembedded (FFPE) samples. The Paradise Reagent System RNAAmplification reagents enable the unprecedented production of microgramquantities of amplified antisense RNA (aRNA) from small quantities oftotal cellular RNA. This proprietary process for linear amplification providesefficient, reproducible results through optimized protocols, reagents, andnucleic acid purification technology using Arcturus’ MiraCol PurificationColumns. FFPE samples with small quantities of total cellular RNA aredifficult to analyze, and standard amplification processes are not designedto accept RNA samples from formalin fixed tissue. The Paradise RNAAmplification reagents can amplify total cellular RNA to generate sufficientaRNA for microarray hybridization experiments. Paradise Reagent SystemRNA Amplification reagents generate unlabeled aRNA, ready for labelingin preparation for microarray experiments or other applications.

B. Performance Specifications

The Paradise Reagent System RNA Amplification reagents are designed toamplify total RNA isolated using the Paradise Extraction/Isolation Reagents.Arcturus recommends the following input of of formalin-fixed RNA:

Minimum: 5 ngRecommended: 10 ng

This amount should yield enough aRNA for duplicate array hybridizations.

C. Quality Control

1. Functional TestingArcturus performs functional testing on each lot of materials usingthe protocol described in this manual.

2. Reagent TestingArcturus tests each lot of enzymes to confirm activity. Buffercomponents must perform correctly under reaction or nucleic acidpurification conditions.

3. Purification Column TestingPurification columns are tested by lot to confirm the absence of nucleicacids and nuclease activity. Column nucleic acid binding and recoveryperformance must meet quality standards.

4. Visual InspectionFinished kits are inspected for proper assembly.

Please read this entire protocol prior to performingamplifications.

RNA Amplification

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D. Storage and Stability

Store the frozen reagent box at –70°C in a non frost-free freezer uponreceipt. Once the reagents are used, storage at -20°C is recommended.The Control RNA vial should be stored at –70°C or belowimmediately upon arrival to ensure maximum stability. Store the roomtemperature box at room temperature. Components stored usingrecommended conditions are guaranteed up to the expiration date on thepackage. For optimal results, using the reagents as soon as possible afterreceipt is recommended.

E. Material Safety and Data Sheets

Material Safety and Data Sheets (MSDS) for kit chemical components areavailable from Arcturus Technical Service or may be downloaded fromwww.arctur.com. Call (888) 446-7911 or (650) 962-3020, or send anemail to [email protected].

3-63-63-63-63-6


II. Components

A. Reagents and Supplies

Component Vial Color Vial#/Name

q 1st Strand Master Mix Red 1q 1st Strand Enzyme Mix Red 2q Enhancer Yellow Eq 1st Strand Nuclease Mix Gold -q 2nd Strand Master Mix White 1q 2nd Strand Enzyme Mix White 2q IVT Buffer Blue 1q IVT Master Mix Blue 2q IVT Enzyme Mix Blue 3q DNase Mix Blue 4q Primer 1 Gray 1q Primer 2 Gray 2q Primer 3 Gray 3

Frozen Reagent Box for KIT0301 and KIT0302

For maximum stability, store Control RNA at –70ºC orbelow.


q DNA Binding Buffer Purple DBq DNA Wash Buffer Purple DWq DNA Elution Buffer Purple DEq RNA Binding Buffer Green RBq RNA Wash Buffer Green RWq RNA Elution Buffer Green REq 0.5 mL Microcentrifuge Tubes --- ---q MiraCol Purification

Columns with collection tubes --- ---

Room Temperature Box


q IVT Buffer Blue 1q IVT Enzyme Blue 2q IVT Master Mix Blue 3q DNase Mix Blue 4q Enhancer Yellow E

Optional Reagents*q aa IVT Master Mix Light Blue AAq Labeling Buffer Light Blue LBq DMSO Light Blue DMSO

2nd Round IVT Box for KIT0302

* Optional reagents are supplied to enable the generation of amino allyl aRNA forhybridization on to sense oligo arrays. See Section V.A Application 3 for details.

RNA Amplification

3-73-73-73-73-7

For maximum stability, store the frozen reagents at –70ºCor below until used. After use, storage at -20ºC isrecommended.



III. Preliminary Notes

A. Recomendations for RNase-free Technique


• Wear disposable gloves and change them frequently.

• After putting on gloves, avoid touching surfaces that may introduceRNases onto the glove surface.

• Do not use reagents not supplied. Substitutions of reagents orcomponents may adversely affect yields or introduce RNases.

• Use only new, sterile RNase-free pipette tips and microcentrifuge tubes.

• Work surfaces should cleaned with commercially available RNasedecontamination solutions prior to performing reactions.

B. Amplified aRNA Contamination

Stray amplified aRNA and cDNA in work area can contaminate precioussamples if the work area is routinely used for performing amplifications. Toensure a work area free of amplified aRNA, please do the following:

1. Irradiate the work area/hood with UV overnight every three to fourdays.

2. Clean surfaces and devices (pipettors, racks, centrifuge, etc.) withcommercially available decontamination solutions everyday or morefrequently depending on use.

C. RNA Preparation

The success of amplification using the Paradise Reagent System RNAAmplification reagents depends on the quality of the source RNA.

Subsequent processing of those samples requires methods that preserveRNA integrity.

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D. RNA Storage

RNA intended for use with the system should be used immediately afterisolation or stored at –70°C or below until use. The control RNA providedwith each Paradise Reagent System RNA Amplification reagents shouldimmediately be stored at –70°C or below upon arrival. Avoid multiplefreeze thaw cycles. Amplified aRNA produced should be used as soon aspossible. Alternatively, the aRNA may be stored at –70°C or below.

E. Additional Equipment and Materials Rquired

1. Equipmento Thermal Cycler with heated lid.o Microcentrifuge for 1.5 mL and 0.5 mL tubes (Eppendorf

5415D or similar)o 0.5 – 10 µL pipettoro 2 – 20 µL pipettoro 20 – 200 µL pipettoro 200 – 1000 µL pipettoro Ice-bath or cold block (4ºC)o Vortex mixer (optional)

2. Materialso 0.5 mL or 0.2 mL RNase-free microcentrifuge tubeso 2 ml lidless tube (PGC Scientific, Cat # 16-8101-06)o Nuclease-free pipette tips

RNA Amplification

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IV. Protocol

A. Overview

The Paradise Reagent System RNA Amplification reagents are optimizedto amplify formalin fixed RNA. The reagents utilize two rounds of a five-step process for linear amplification of the mRNA fraction of total cellularRNA: (a) first-strand synthesis reaction that yields cDNA incorporating aT7 promoter sequence; (b) second-strand synthesis reaction utilizingexogenous primers that yields double-stranded cDNA; (c) cDNApurification using specially designed MiraCol Purification Columns;(d) in vitro transcription (IVT) utilizing T7 RNA polymerase yieldsantisense RNA (aRNA); and (e) aRNA isolation with the MiraColPurification Columns. KIT0302 entails two rounds of amplification foreach sample. KIT0301 entails one round of amplification followed bycDNA synthesis. This ds cDNA is the template to be used in a commerciallyavailable transcript labeling kit. To save time, in vitro transcription may beperformed overnight with the proper thermal cycler programming.

3-103-103-103-103-10

* Optional reagents are supplied to enable the generation of amino allyl aRNA for hybridization on to sense oligo arrays.See Section V.A Application 3 for details.


B. Thermal Cycler Programming

Thermal cyclers provide a convenient and reproducible method ofincubating reactions according to specified temperatures and times in theprotocol. A thermal cycler program for use appears on page 3-12. Theprogram is not intended for automatic progression from one time andtemperature set to another. The program lists a 4°C hold after eachincubation or incubation cycle when it is necessary to remove the reactionsfrom the thermal cycler to add reagents. After the addition of reagents,place the sample back into the thermal cycler and resume the program.

C. Time Requirements

The table below presents typical time requirements for completion of theprotocol. Times reflect total handling and reaction times of each step. Notethat there are safe stopping points for pausing the amplification process,and the times presented reflect a continuous, uninterrupted process.

Using a thermal cycler with a heated lid is important. Theheated lid ensures proper temperature distribution withinthe reaction tube and prevents evaporative condensationthat alters the reaction mixture concentrations.

1st Round 2nd RoundSteps (hours) (hours)

1st Strand Synthesis 3.0 2.02nd Strand Synthesis 1.0 1.0cDNA Purification 0.5 0.5Total (before IVT) 4.5 3.5In Vitro Transcription 8.5 *variesaRNA Purification 0.5 0.5Total 13.5 *varies

Time Required (KIT0301)

* KIT0301 time requirement will depend on the commercially availabletranscript labeling kit used.

RNA Amplification

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1st Round 2nd RoundSteps (hours) (hours)

1st Strand Synthesis 3.0 2.02nd Strand Synthesis 1.0 1.0cDNA Purification 0.5 0.5Total (before IVT) 4.5 3.5In Vitro Transcription 8.5 8.5aRNA Purification 0.5 0.5Total* 13.5 12.5

Time Required (KIT0302)

* For samples processed using the optional IVT Master Mix, an additional 2.5hours will be requied for amino-allyl aRNA labeling.



IVT

Program

oC70 1 hour4 hold

42 1.5 hours4 hold

37 30 minutes95 5 minutes4 hold

95 2 minutes4 hold

25 10 minutes37 30 minutes70 5 minutes4 hold

42 8 hours4 hold (optional overnight hold)

37 15 minutes4 hold

Round One

1st Strand Synthesis

2nd Strand Synthesis

IVT

Round Two

oC70 5 minutes

4 Hold25 10 minutes37 1.5 hours

4 Hold95 5 minutes

4 Hold37 30 minutes70 5 minutes

4 Hold42 8 hours

4 Hold (optional overnight hold)37 15 minutes

4 Hold

1st Strand Synthesis

2nd Strand Synthesis

The 4°C steps in the thermal cycler program allow for bufferand reagent addition and mixing steps at certain pointsduring the amplification process and are not intended forindefinite hold unless noted.

Do not allow incubation times and temperatures to deviatefrom the protocol.

The IVT step is for KIT0302 only.

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D. Detailed Protocol

1. Protocol Notes

a. When adding reagent to samples or master mixes, pipette mixturesup and down several times to ensure complete transfer of reagentfrom the pipette tip.

b. Prior to the first use of an enzyme, gently mix (do not vortex) andbriefly microcentrifuge the vial to ensure that all enzyme is mixedand collected at the bottom of the vial. Enzyme may collect on thevial wall or cap during shipment.

c. Keep thawed reagents and reaction tubes in cold blocks at 4°C whileadding reagents to samples.

d. Prior to each incubation, mix samples thoroughly by flicking thereaction tube (unless noted in protocol) to ensure process performance.Spin down before proceeding. DO NOT VORTEX REACTIONSAMPLES.

e. Use a microcentrifuge to spin down all components and samplesfollowing each mixing step.

f. Clean all amplification process equipment with an RNase eliminatorsuch as RNase AWAY (Life Technologies) to minimize the risk ofRNase contamination.

g. During enzyme and buffer dispensing, keep the reaction tubewith sample on ice or chilled in a 4°C cold block. Do not freezesamples unless it is indicated to be safe to do so in the protocol.

2. Sample and Reagents Preparation

a. Thaw frozen kit components, as needed, and mix with gentle vortexingor by inverting the tubes several times, spin down, and place on ice.When enzyme mixtures must be removed from –20°C storage foruse, always keep them in a cold block or in an ice bucket at the labbench.

b. Allow In Vitro Transcription (IVT) Buffer (Blue-labeled Vial 1),Master Mix (Blue-labeled Vial 2) and Enhancer (Yellow-labeled Vial)to assume room temperature (22–25°C), and mix by inverting orflicking the tube. Spin down if necessary. Dissolve all visible solidsprior to use.

c. The Paradise Reagent System RNA Amplification reagents areoptimized for the input of formalin modified total cellular RNA.

d. Although excess enzyme and reagents are provided in all vials, there isinsufficient volume to prepare extra reactions.

e. Two IVT Master Mix reagents are provided with KIT0302. ConsultAppendix A to determine which vial to use for the intendedapplication.

When making master mixes, use only 10% overage per sampleto avoid running out of reagent.

RNA Amplification

3-133-133-133-133-13



3. Nucleic Acid Elution Using Spin Columns

Spin columns and 0.5 ml microcentrifuge tubes are provided for nucleicacid elution. Improper orientation of tubes during centrifugation mayresult in cap breakage or sample loss.

To correctly use the column-tube assembly, insert a spin column into the0.5 ml tube, aligning the two cap hinges as illustrated. Load ElutionBuffer onto the column and incubate as directed. Place the column-tubeassembly into a 2 ml lidless support tube (PGC Scientific, Catalog #16-8101-06) in the centrifuge rotor; alternately, retain and reuse the 2 mllidless collection tubes provided. (Some varieties of 2 ml tubes will notprovide enough support. Contact Arcturus Technical Support for otheralternatives.) Skip one rotor position between assemblies, and positionassemblies with the 0.5 ml tube cap trailing the tube during centrifugationas shown. (Check for a mark on the centrifuge indicating rotation direction.)Centrifuge as directed in the protocol.

4. RNA Input Recommendations

Arcturus recommends the following input of of formalin-fixed RNA:Minimum: 5 ngRecommended: 10 ng

5. Control Amplifications

A control RNA sample is provided along with each kit to be used as acontrol template to verify amplification efficacy. Use 10 µL of this RNAfor control amplifications. 10 µL of this RNA contains 5 ng of total RNA.50 µL of control RNA is provided.

6. Work Space Recommendations

Due to the ultra sensitivity of the reagents, it is very important to preventRNA, DNA, and nuclease contamination. Work surfaces should be cleanedbefore and after each use. Perform all dispensing in a work hood that hasbeen irradiated with UV to remove contaminants from previousamplification experiments.

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7. Protocol

Round One: 1st Strand Synthesis

1. Prepare RNA sample in a total volume of 10 –11 µL in a 0.5 mLRNase-free microcentrifuge tube. For recommended sample inputquantities, refer to page 3-14.

2. Thaw Primer 1 (Gray-labeled Vial-1), thoroughly mix, and spin down.

3. Into 10 –11 µl RNA sample, add 1.0 µL of Primer 1, mix thoroughlyby flicking the tube and spin down.

4. Incubate at 70°C for 1 hour then chill the samples to 4°C for at leastone minute. Hold the sample at 4°C until ready to proceed. Spindown the contents before proceeding to the next step. Keep sampleson cold block at 4°C.

5. Place 1st Strand Master Mix (Red-labeled Vial-1) on ice. 1st StrandMaster Mix and Enhancer (Yellow-labeled Vial) must be thawed,thoroughly mixed with all solids dissolved, and maintained at 4°Cuntil used. 1st Strand Enzyme Mix does not require thawing and canbe placed directly on ice. Mix enzyme throughly by inverting severaltimes. Spin briefly.

6. Add 1st Strand Synthesis components in the order listed in thefollowing table. If you are performing several amplifications, you maywish to prepare a Complete 1st Strand Synthesis Mix based on thefollowing table, and add 9.0 µL Complete 1st Strand Synthesis Mixto each sample. Mix thoroughly by flicking the tube and spindown.DO NOT VORTEX. Use only 10% overage for each sample toavoid running out of reagents.

Read Sections B, C and D1-D6 prior to beginning.

Place components back onto ice or refreeze immediately afterdispensing the reagent. Do not leave reagents at roomtemperature.

Gray

Red

7. Incubate at 42°C for 1.5 hours then chill the sample to 4°C for atleast one minute. Do not hold at 4°C over a prolonged period of time.

8. Thoroughly mix and spin down 1st Strand Nuclease Mix (Gold-labeled Vial). Place on ice.

9. Add 2.0 µL of 1st Strand Nuclease Mix to the sample, mix thoroughlyby flicking the tube and spin down.

10. Incubate the sample at 37°C for 30 minutes followed by 95°C forfive minutes.

11. (Optional) You may remove a 2.0 µL sample at this point in theprotocol to assess the integrity of the starting mRNA by QuantitativeReal-Time PCR (Q-RT-PCR.)

12. Chill the sample to 4°C for at least one minute and hold at thattemperature until ready to proceed.

Red

Complete 1st Strand Synthesis Mix

Component Amount Vial #

Enhancer 2 µL Yellow1st Strand Master Mix 5 µL Red-11st Strand Enzyme Mix 2 µL Red-2Complete 1st Strand 9 µLSynthesis Mix

It is okay to stop at this point in the protocol. Sample may be stored at–20°C overnight.

Gold

Yellow

Example: Amount for 8 reactions

Enhancer 2 x 8.8 17.6 µL1st Strand Master Mix 5 x 8.8 44.0 µL1st Strand Enzyme Mix 2 x 8.8 17.6 µLComplete 1st Strand 79.2 µLSynthesis Mix

RNA Amplification

3-153-153-153-153-15



1. Thaw Primer 2 (Gray-labeled Vial - 2), thoroughly mix, and spindown.

2. Add 1.0 µL of Primer 2 at 4°C. Mix thoroughly by flicking the tubeand spin down.

3. Incubate sample at 95°C for 2 minutes, then chill and maintain thesample at 4°C for at least 1 minute.

4. Place 2nd Strand Components (White-labeled Vials) on ice. 2ndStrand master mix must be thawed, thoroughly mixed with all solidsdissolved, and maintained at 4°C until used. 2nd Strand EnzymeMix does not require thawing and can be placed directly on ice. Mixenzyme thoroughly by inverting several times. Spin briefly.

5. Add 2nd Strand Synthesis components separately in the order listedin the following table. If you are performing several amplifications,you may wish to prepare a Complete 2nd Strand Synthesis Mix basedon the following table, and add 30 µL Complete 2nd Strand SynthesisMix to each sample. Mix thoroughly by flicking the tube and spindown.

Round One: 2nd Strand Synthesis

6. Incubate the sample as follows:• 25°C 10 minutes

• 37°C 30 minutes

• 70°C 5 minutes

• 4°C Hold until ready to proceed (up to a maximum of 30 minutes)

Place components back onto ice or refreeze immediately afterdispensing the reagent. Do not leave reagents at roomtemperature for any extended period of time.

White

White

Component Amount White Vial #

2nd Strand Master Mix 29 µL 12nd Strand Enzyme Mix 1 µL 2Complete 2nd Strand 30 µLSynthesis Mix(Store at 4oC until use.)

Complete 2nd Strand Synthesis Mix

Gray


2nd Strand Master Mix 29 x 8.8 255.2 µL2nd Strand Enzyme Mix 1x 8.8 8.8 µLComplete 2nd Strand 264 µLSynthesis Mix

3-163-163-163-163-16


1. Add 250 µL of DNA Binding Buffer (DB) to a new purificationcolumn seated in the collection tube provided. Incubate for fiveminutes at room temperature. Centrifuge at 16,000 x g for one minute.

2. Add 200 µL of DB to the 2nd Strand Synthesis sample tube, mixwell, and pipette the entire volume into the purification column.

3. To bind cDNA, centrifuge at 100 x g (or lowest speed setting available)for two minutes, immediately followed by a centrifugation at 10,000x g for 1 minute to remove flowthrough.

4. Add 250 µL of DNA Wash Buffer (DW) to the column and centrifugeat 16,000 x g for two minutes. Check the purification column forany residual wash buffer. If any wash buffer remains, re-centrifuge at16,000 x g for one minute.

5. Discard the collection tube and flowthrough.

6. Place the column into the provided 0.5 mL microcentrifuge tubeand carefully add 11 µµµµµL of DNA Elution Buffer (DE) onto thecenter of the purification column membrane. Gently touch the tip ofthe pipette to the surface of the membrane while dispensing DE toensure maximum absorption of DE into the membrane. Gently tapthe purification column to distribute the buffer, if necessary.

7. Incubate for one minute at room temperature.

8. Place each column-tube assembly into the 2 ml support tube in therotor with the 0.5 ml tube cap trailingthe tube.

9. Centrifuge at 1000 x g for one minute, followed immediately by16,000 x g for one minute. Discard the column and retain the elutioncontaining the cDNA.

Round One: cDNA Purification

Purple

DNA Binding Buffer (DB) must be at room temperatureand thoroughly mixed before use. A precipitate may formduring long term storage. Dissolve precipitate prior to use bymixing. If necessary, warm the DB vial to redissolve.

Purple

Purple

Tubes must be properly oriented in the rotor during elution.See Section IV.D.3 for details.It is safe to stop at this point in the protocol. You may store the sample

overnight at –20°C.

Avoid splashing flowthrough in the collection tube onto thecolumn. If flowthrough waste liquid wets the outside of thepurification column, recentrifuge the column at16,000 x gto remove liquid.

RNA Amplification

3-173-173-173-173-17



Round One: In Vitro Transcription (IVT)

1. Thaw IVT Buffer (Blue-labeled Vial 1), Master Mix (Blue-labeledVial 2) and Enhancer (Yellow-labeled Vial) to room temperature(22 – 25°C) and thoroughly mix to dissolve all solids. IVT EnzymeMix does not require thawing and can be put directly on ice. Mixenzyme thoroughly by inverting several times. Spin briefly.

2. Add IVT components in the order listed in the following table. Ifyou are performing several amplifications, you may wish to prepare aComplete IVT Reaction. Mix according to the following table, andadd 12 µL Complete IVT Reaction Mix to each sample. Mixthoroughly by flicking the tube and spin down.

3. Incubate at 42°C for 8 hours. Chill the sample(s) to 4°C.

Blue Blue

Blue

Place components back onto ice or re-freeze immediately afterdispensing the reagent. Do not leave reagents at roomtemperature.

Blue

DNase Mix must be thoroughly mixed and held at 4°Cuntil used.

RNA may be adversely affected if not purified immediatelyafter DNase treatment.

At this point in the protocol, you may hold the reaction mixture at 4°Cin the thermal cycler overnight.

4. Add 1 µL DNase Mix (Blue-labeled Vial 4). Mix thoroughly andspin down. Incubate at 37°C for 15 minutes. Chill the sample(s) to4°C. Proceed immediately to aRNA purification.


Enhancer 2 µL YellowIVT Buffer 2 µL Blue-1IVT Master Mix 6 µL Blue-2IVT Enzyme Mix 2 µL Blue-3Complete IVTReaction Mix 12 µL

Complete IVT Reaction MixYellow

Example: IVT Reaction Mix for 8 reactions

Enhancer 2 x 8.8 17.6 µLIVT Buffer 2 x 8.8 17.6 µLIVT Master Mix 6 x 8.8 52.8 µLIVT Enzyme Mix 2 x 8.8 17.6 µLComplete IVT Reaction Mix 105.6µL

3-183-183-183-183-18


Round One: Antisense RNA (aRNA) Purification

1. Add 250 µL of RNA Binding Buffer (RB) to a new purificationcolumn and incubate for five minutes at room temperature. Centrifugeat 16,000 x g for one minute.

2. Add 120 µL of RB to the IVT reaction sample and mix thoroughly.Pipette the entire volume into the purification column.

3. To bind aRNA, centrifuge at 100 x g (or lowest speed setting available)for two minutes, immediately followed by a centrifugation at 10,000x g for one minute to remove flowthrough.

4. Add 200 µL of RNA Wash Buffer (RW) to the purification columnand centrifuge at 10,000 x g for one minute.

5. Add 200 µL of fresh RW to the purification column, and centrifugeat 16,000 x g for two minutes. Check the purification column for anyresidual wash buffer. If any wash buffer remains, re-centrifuge at16,000 x g for one minute.


7. Place the purification column into a new 0.5 mL microcentrifugetube provided in the kit and carefully add 12 µµµµµL of RNA ElutionBuffer (RE) directly to the center of the purification column membrane.Gently touch the tip of the pipette to the surface of the membranewhile dispensing RE to ensure maximum absorption of RE into themembrane. Gently tap the purification column to distribute the buffer,if necessary.

8. Incubate at room temperature for one minute.

9. Place each column-tube assembly into the 2 ml support tube in rotorwith the 0.5 ml tube cap trailing the tube.

10. Centrifuge at 1,000 x g for one minute, immediately followed by16,000 x g for one minute. Discard the purification column andretain the elution containing the aRNA.

11. Generate labeled aRNA in Round Two or store the purified aRNA at–70°C overnight.

Green

RNA Binding Buffer (RB) must be at room temperature andthoroughly mixed before use. A Precipitate may form duringlong term storage. Dissolve precipitate prior to use by mixing.If necessary, warm the RB vial to redissolve.

Green

Avoid splashing flowthrough in the collection tube onto thepurification column. If flowthrough waste liquid wets theoutside of the purification column, recentrifuge the columnat 16,000 x g to remove the liquid.

Green

Tubes must be properly oriented in the rotor during elution.See Section IV.D.3 for details.

End of Round One

RNA Amplification

3-193-193-193-193-19



Round Two: 1st Strand Synthesis

1. Thaw Primer 2 (Gray-labeled Vial 2), thoroughly mix, and spindown.

2. Into eluted aRNA product from Round One, add 1.0 µL of Primer 2,mix thoroughly by flicking the tube and spin down.

3. Incubate the microcentrifuge tube at 70°C for 5 minutes then chillthe samples to 4°C for at least 1 minute.

4. Place 1st Strand Synthesis components (Red-labeled Vials) on ice.1st Strand Master Mix and Enhancer (Yellow-labeled Vial) must bethawed, thoroughly mixed with all solids dissolved, and maintainedat 4°C until used. 1st Strand Enzyme Mix does not require thawingand can be placed directly on ice. Mix enzyme thoroughly by invertingseveral times. Spin briefly.

5. Add 1st Strand Synthesis components separately in the order listed inthe following table. If you are performing several amplifications, youmay wish to prepare a Complete 1st Strand Synthesis Mix based onthe following table, and add 9.0 µL Complete 1st Strand SynthesisMix to each sample. Mix thoroughly by flicking the tube and spindown. DO NOT VORTEX. Use only 10% per reaction overage toavoid running out of reagents.

It is okay to stop at this point in the protocol. Sample(s) may be storedovernight at –20°C.

Red


Red


Enhancer 2 µL Yellow1st Strand Master Mix 5 µL Red-11st Strand Enzyme Mix 2 µL Red-2Complete 1st Strand Synthesis Mix 9 µL

Complete 1st Strand Synthesis Mix

Gray

Yellow


Enhancer 2 x 8.8 17.6 µL1st Strand Master Mix 5 x 8.8 44.0 µL1st Strand Enzyme Mix 2 x 8.8 17.6 µLComplete 1st Strand 79.2 µLSynthesis Mix

3-203-203-203-203-20

6. Incubate the sample(s) at 25°C for 10 minutes then at 37°C for 1.5hours.

7. Chill the sample(s) to 4°C for at least one minute.


1. Thaw Primer 3 (Gray-labeled Vial 3), thoroughly mix, and spindown.

2. Add 1.0 µL of Primer 3 to the sample at 4°C. Mix thoroughly andspin down.

3. Heat the sample(s) at 95°C for five minutes then cool sample(s) to4°C for at least one minute.

4. Place 2nd Strand Components (White-labeled Vials) on ice. The2nd Strand Master Mix must be thawed, thoroughly mixed with allsolids dissolved, and maintained at 4°C until used. 2ndStrand EnzymeMix does not require thawing and can be placed directly on ice. Mixenzyme thoroughly by inverting several times. Spin briefly.

5. Add 2nd Strand Synthesis components separately in the order listedin the following table. If you are performing several amplifications,you may wish to prepare a Complete 2nd Strand Synthesis Mixbased on the following table, and add 30 µL Complete 2nd StrandSynthesis Mix to each sample. Mix thoroughly by flicking the tubeand spin down.

Round Two: 2nd Strand Synthesis

Gray


White

White

Component Amount White Vial #

2nd Strand Master Mix 29 µL 12nd Strand Enzyme Mix 1 µL 2Complete 2nd Strand Synthesis Mix 30 µL(Store at 4°C until use)

Complete 2nd Strand Synthesis Mix Example: Amount for 8 reactions

2nd Strand Master Mix 29 x 8.8 255.2 µL2nd Strand Enzyme Mix 1x 8.8 8.8 µLComplete 2nd Strand 264 µLSynthesis Mix

RNA Amplification

3-213-213-213-213-21

6. Incubate the sample(s) as follows:· 37°C 30 minutes· 70°C 5 minutes· 4°C Hold until ready to proceed (up to a maximum of 30

minutes)



3. To bind cDNA, centrifuge at 100 x g (or lowest speed setting available)for two minutes, immediately followed by a centrifugation at 10,000x g for 1 minute to remove flowthrough.

4. Add 250 µL of DNA Wash Buffer (DW) to the column and centrifugeat 16,000 x g for two minutes. Check the purification column forany residual wash buffer. If any wash buffer remains, recentrifuge at16,000 x g for one minute.


6. Place the column into the provided 0.5 mL microcentrifuge tubeand carefully add 11 µµµµµL of DNA Elution Buffer (DE) onto thecenter of the purification column membrane. Gently touch the tip ofthe pipette to the surface of the membrane while dispensing DE toensure maximum absorption of DE into the membrane. Gently tapthe purification column to distribute the buffer, if necessary.


8. Place each column-tube assembly into the 2 ml support tube in therotor with the 0.5 ml tube cap trailing the tube.

9. Centrifuge at 1000 x g for one minute, followed immediately by16,000 x g for one minute. Discard the column and retain theelution containing the cDNA.

Round Two: cDNA Purification

It is okay to stop at this point in the protocol. Sample may be storedovernight at –20°C.

Purple

DNA Binding Buffer (DB) must be at room temperatureand thoroughly mixed before use. A precipitate may formduring long-term storage. Dissolve precipitate by mixing. Ifnecessary, warm the DB vial to redissolve.

Purple

Avoid splashing flowthrough in the collection tube onto thepurification column. If flowthrough waste liquid wets theoutside of the purification column, recentrifuge at 16,000 xg to remove liquid.

Purple


1. Add 250 µL of DNA Binding Buffer (DB) to a new purificationcolumn seated in the collection tube provided. Incubate for fiveminutes at room temperature. Centrifuge at 16,000 x g for one minute.

2. Add 200 µL of DB to the 2nd Strand Synthesis sample tube, mixwell, and pipette the entire volume into the purification column.

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KIT0301: Paradise Reagent System 1.5 Rounds of Amplification

KIT0301: Paradise Reagent System 1.5 Rounds of AmplificationProceed to KIT0301 Section below.

KIT0302: Paradise Reagent System 2 Rounds of AmplificationProceed to KIT0302 Section on page 3-25.

Round Two Amplification

Use the reagents and protocols of the commercially available transcriptlabeling kit. Incorporation of label may be accomplished for a biotin label,for example, with the ENZO BioArray HighYield RNA Transcript LabelingKit (Affymetrix). Thoroughly read the instruction manual prior to use.After transcript labeling is complete, proceed to aRNA purification topurify the labeled aRNA. The protocol assumes a volume of 40 µl oflabeled aRNA.

1. Add 250 µL of RNA Binding Buffer (RB) to a new purificationcolumn seated in the collection tube provided. Incubate for fiveminutes at room temperature. Centrifuge at 16,000 x g for oneminute.


3. To bind aRNA, centrifuge at 100 x g (or lowest speed setting available)for two minutes, immediately followed by a centrifugation at 10,000x g for 1 minute.


5. Add 200 µL of fresh RW to the purification column, and centrifugeat 16,000 x g for two minutes. Check the column for any residualwash buffer. If any wash buffer remains, recentrifuge at 16,000 x g forone minute.


Green

RNA Binding Buffer (RB) must be at room temperature andthoroughly mixed before use. A precipitate may form duringlong-term storage. Dissolve precipitate by mixing. If necessary,warm the RB vial to redissolve.

Green

Avoid splashing flowthrough in the collection tube onto thepurification column. If flowthrough waste liquid wets theoutside of the column, re-centrifuge the column at 16,000 x gto remove the liquid.

KIT0301: Round Two Antisense RNA (aRNA) Purification

RNA Amplification

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7. Place the purification column into a new 0.5 mL microcentrifugetube provided in the Kit and carefully add 30 µµµµµL of RNA ElutionBuffer (RE) directly to the center of the purification columnmembrane. Gently touch the tip of the pipette to the surface of themembrane while dispensing RE to ensure maximum absorption ofRE into the membrane. Gently tap the purification column todistribute the buffer, if necessary.



10. Centrifuge at 1000 x g for one minute, followed immediately by16,000 x g for one minute. Discard the column and retain the elutioncontaining the aRNA.

11. Measure the O.D. of the product at A260

and A280

(Appendix B).

12. Analyze the aRNA with the Agilent Bioanalyzer (Appendix D) or bygel electrophoresis (Appendix E).

KIT0301: End of Round Two

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1. Thaw IVT Buffer (Blue-labeled Vial 1), Master Mix (Blue-labeledVial 2) and Enhancer (Yellow-labeled Vial) to room temperature(22 – 25°C) and thoroughly mix to dissolve all solids. IVT EnzymeMix does not require thawing and can be put directly on ice. Mixenzyme thoroughly by inverting several times. Spin briefly.

2. Add IVT components in the order listed in the following table. Ifyou are performing several amplifications, you may wish to prepare aComplete IVT Reaction Mix according to the following table, andadd 12 µl Complete IVT Reaction Mix to each sample. Mixthoroughly by flicking the tube and spin down.

At this point in the protocol, you may hold the reaction mixture at 4°Cin the thermal cycler overnight.

Blue Blue

Blue


Blue

DNase Mix must be thoroughly mixed and held at 4°Cuntil used.

RNA may be adversely affected if not purified immediatelyafter DNase treatment.

Yellow


Enhancer 2 µL YellowIVT Buffer 2 µL Blue-1IVT Master Mix* 6 µL Blue-2IVT Enzyme Mix 2 µL Blue-3Complete IVT Reaction Mix 12 µL

Complete IVT Reaction Mix

*Two IVT Master Mix vials are provided with the Paradise RNA Amplificationreagents. Consult the Appendix A to determine which vial to use for the intendedapplication.

or

Light Blue

Example: IVT Reaction Mix for 8 reactions

Enhancer 2 x 8.8 17.6 µLIVT Buffer 2 x 8.8 17.6 µLIVT Master Mix 6 x 8.8 52.8 µLIVT Enzyme Mix 2 x 8.8 17.6 µLComplete IVT Reaction Mix 105.6µL

3. Incubate at 42°C for 8 hours. Chill the sample(s) to 4°C.

Round Two: In Vitro Transcription (IVT)

4. Add 1 µL DNase Mix (Blue-labeled Vial 4). Mix thoroughly andspin down. Incubate at 37ºC for 15 minutes. Chill the sample(s) to4ºC. Proceed immediately to aRNA purification.

RNA Amplification

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KIT0302: Paradise Reagent System 2 Rounds of Amplification



1. Add 250 µL of RNA Binding Buffer (RB) to a new purificationcolumn seated in the collection tube provided. Incubate for fiveminutes at room temperature. Centrifuge at 16,000 x g for oneminute.


3. To bind aRNA, centrifuge at 100 x g (or lowest speed setting available)for two minutes, immediately followed by a centrifugation at 10,000x g for 1 minute.


5. Add 200 µL of fresh RW to the purification column, and centrifugeat 16,000 x g for two minutes. Check the column for any residualwash buffer. If any wash buffer remains, recentrifuge at 16,000 x g forone minute.


7. Place the purification column into a new 0.5 mL microcentrifugetube provided in the Kit and carefully add 30 µ µ µ µ µL of RNA ElutionBuffer (RE) directly to the center of the purification column membrane.Gently touch the tip of the pipette to the surface of the membrane



10. Centrifuge at 1000 x g for one minute, followed immediately by16,000 x g for one minute. Discard the column and retain the elutioncontaining the aRNA.


and A280

(Appendix B).

12. Analyze the aRNA with the Agilent Bioanalyzer (Appendix D) or bygel electrophoresis (Appendix E).

Green

RNA Binding Buffer (RB) must be at room temperature andthoroughly mixed before use. A precipitate may form duringlong-term storage. Dissolve precipitate by mixing. If necessary,warm the RB vial to redissolve.

Green

Avoid splashing flowthrough in the collection tube onto thepurification column. If flowthrough waste liquid wets theoutside of the column, re-centrifuge the column at 16,000 x gto remove the liquid.

KIT0302: Round Two Antisense RNA (aRNA) Purification


KIT0302: End of Round Two

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Green


V. Appendices

A. Applications of aRNA

The Paradise Reagent System can be used to yield a suitable labeled RNAsample for hybridization to nucleic acid in a variety of formats. The RNAsample may be labeled in a number of different ways, including those listedbelow.

Application 1: Direct mRNA Fluorescent Labeling:

After analysis of the aRNA with the Agilent BioAnalyzer (Appendix C) orby gel electrophoresis (Appendix D) as described in Round Two: AntisenseRNA (aRNA) Purification (see Step 12), aRNA may be directly labeledwith a fluroescent marker. Direct mRNA labeling can be accomplishedusing commercially available reagent kits, including:a. CyScribe Direct™ mRNA Labelling Kit available from Amersham.b. ULYSIS® Nucleic Acid Labeling Kits available from Molecular Probes.c. MICROMAX ASAP RNA Labeling Kit available from Perkin Elmer.

Application 2: Direct cDNA Cy3 or Cy5 Fluorescent Labeling(For use with KIT0302)

The protocol described here may be used to prepare Cyanine 3- or Cyanine5-labeled cDNA from aRNA generated using the Paradise Reagent SystemRNA Amplification Kit for hybridization to cDNA microarrays. Thisprotocol provides labeled probe of sense orientation from 5-10 microgramsof aRNA, a sufficient quantity for replicate hybridizations on cDNAmicroarrays. However, such probes are typically not used for oligonucleotidearrays, since the targets on such arrays are also generally in the senseorientation.

Reagents Used Maker Catalog#RNase AWAY® Invitrogen 10328-011Cy3 labeled dUTP Amersham PA53022Cy5 labeled dUTP Amersham PA55022RNAsin® Ribonuclease Inhibitor Promega N2515SuperScript II RT and Buffer Invitrogen 18064-071Nuclease Free Water Invitrogen 10977-023Rnase H Invitrogen 18021-071Random Hexamer Operon custom-madeQiaQuick® PCR Purification Kit Qiagen® 28106

RNA Amplification

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Method

1. Take 5-10 µg of amplified aRNA and adjust the volume to 22 µl withnuclease-free water.

2. Add 2 µl of 5 mg/ml random hexamer.

3. Mix well by flicking, then briefly spin down by centrifugation.

4. Heat the tube to 70°C for 10 minutes, then 4OC for 2 minutes in thethermal cycler.

5. During incubation, prepare the first-strand master mix as describedbelow.

If the volume is greater than 22 µµµµµl, concentrate it down to22 µµµµµl using a vacuum concentrator, paying attention to notcompletely dry down the aRNA sample.

6. When incubation is complete, mix the tube well by flicking, and thenbriefly spin down by centrifugation.

7. Add 26 µl of the above first stand master mix to each reaction tube.

8. Mix well by flicking, and then briefly spin down by centrifugation.

9. Incubate at 27°C for 10 minutes, followed by 37°C for 2 hours in thethermal cycler.

10. Treat with 2 units of RNase H for 20 minutes at 37°C in the thermalcycler.

11. Immediately proceed to PCR product purification usingQiaQuick®PCR Purification Kit. Pre-treat the columns placed incollection tube by incubating 100 µl of QiaQuick PB buffer for 5minutes, and then centrifuge at 13200 rpm (or full speed on a 5415CEppendorf Centrifuge) for 1 minute.

12. Add 260 µl of QiaQuick® PB buffer to the sample tube.

13. Mix well by flicking, and then briefly spin down by centrifugation.

14. Load the sample onto the pre-treated columns. Centrifuge at 6000rpm for 1 minute.

First Strand Master Mix - 1xFirst Strand Buffer 10 µl0.1 M DTT 5 µl25 mM dNTP 1 µl1 mM dUTP-Cy3 or Cy5 2 µl1 mM dTTP 2 µlRNasin® 2 µlSuperscript II RT 4 µlTotal 26 µµµµµl

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RNA Amplification

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15. Discard flowthrough. Place the column into the same collection tube.

16. Wash with 750 µl of QiaQuick PE buffer. Centrifuge at 13200 rpmfor 1 minute.

17. Discard flowthrough. Place the column back into the same collectiontube.

18. Centrifuge at 13200 rpm for an additional 2 minutes to removeresidual wash solution.

19. Place the column into a clean 2 ml microcentrifuge tube.

20. Add 50 µl of nuclease-free water, pH 8.5, directly onto the columnmembrane. Incubate for 3–5 minutes, and then centrifuge atmaximum speed for 1 minute.

21. If the column still shows residual probe, add another 30 µl of nucleasefree water, pH 8.5, directly onto the column membrane, incubate for1minute, and centrifuge at maximum speed for 1 minute.

After centrifuging, make sure that the entire sample has passedthrough the column, and that the column is completely dry. Ifnot, centrifuge for an additional 1 minute at 6000 rpm.When dry, the column will be visibly pink (Cy3) or blue(Cy5) if the reaction was successful.

If the labeling reaction was successful, the eluate should bevisibly pink (Cy3) or blue (Cy5) in color.



Application 3: Amino-allyl aRNA Labeling

This protocol is intended for use with amino-allyl modified aRNA whichwas generated using the optional IVT Master Mix components.

Reagent Maker Catalog#

Cy3 monoReactive Dye Amersham PA23001

Cy5 monoReactive Dye Amersham PA25001

Alexa Fluor® 555 reactive Molecular Probes A-32756dye decapack *for microarrays*

Alexa Fluor® 647 reactive dye Molecular Probes A-32757decapack *for microarrays*

Alexa Fluor® 555 and Alexa Molecular Probes A-32755Fluor® 647 reactive dye decapacks*for microarrays* *set of 2 x 10 vials**includes A-32756 and A-32757decapacks*

Protocol

Labeling Reaction:

Resuspend 1mg monoreactive dye in 50 µl of DMSO. Save unused vialsin the dark at 2–6ºC.

1. Take 15 µg of amino-allyl aRNA in 7.5 µl of nuclease free water.a. Sample should be maintained on a cold block.

2. Add 2.5 µl of Labeling Buffer (LB) to the sample.

3. Add 10 µl of the resuspended dye into 10 µl of the sample.

4. Mix thoroughly by flicking the tube. Spin down briefly.

5. Incubate at room temperature in the dark for 1 hour.

6. Proceed directly to purification of labeled aRNA.

aRNA Purification:

1. Pre-treat column by adding 250 µl of RNA Binding Buffer (RB) toa new purification column. Incubate the column at room temperaturefor 5 minutes. Centrifuge at 16,000 x g for one minute.

2. Add 225 µl of RB to the transcript labeling reaction sample and mixthoroughly. Pipette the entire sample volume into the purificationcolumn.

3. Centrifuge at 100 x g (or lowest speed setting available) for 2 minutes,immediately followed by a centrifugation at 10,000 x g for 1 minute.

Do not use re-suspended dye that is over 2 days old. DMSOis hygroscopic. Store tightly capped.

Do not allow the samples to incubate longer than 1 hour.

Use reagents supplied in the Labeling Purification Regaentsbox.

To obtain 15 µg of aRNA in 7.5µl, you may dry down15µg of aRNA and resuspend in 7.5 µl of nuclease free water,or concentrate the aRNA to 2 µg /µl and use 7.5 µl of thesample.

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4. Discard flowthrough. Place the column into the same collectiontube.

5. Add 250 µl of RNA Wash Buffer (RW) to the purification columnand centrifuge at 10,000 x g for 1 minute.

6. Repeat Step 5.

7. Add 250 µl of fresh RW to the column and centrifuge at 16,000 x g(full speed) for 2 minutes. Check the purification column for anyresidual wash buffer. If any wash buffer remains, re-centrifuge at16,000 x g for one minute.


9. Place the purification column into a new 0.5 mL microcentrifugetube provided in the kit and carefully add 50 µl of RNA elutionBuffer (RE) directly onto the center of the purification columnmembrane. Gently touch the tip of the pipette to the surface of themembrane while dispensing RE to ensure maximum absorption ofRE into the membrane. Gently tap the purification column todistribute the buffer if necessary.

10. Incubate at room temperature for one minute.

11. Place the assembly into the 2 ml support tube in the rotor with the0.5 ml tube cap trailing the tube.

12. Centrifuge at 1000 x g for one minute, immediately followed by16,000 x g for one minute. Discard the purification column andretain the elution containing the labeled aRNA.


, A280

and A550

/A650

todetermine the yield and frequency of incorporation (FOI) by makinga dilution of 1:10 (5 µl sample + 45 µl nuclease free water).

14. Store any remaining samples at –80ºC until ready for hybridization.

Application 4: Immobilization of Amino-allyl Modified aRNA

The Paradise Reagent System can be used to yield a suitable labeled RNAsample for immobilization on a solid support. This protocol is intendedfor use with amino-allyl modified aRNA which was generated using theoptional IVT Master Mix components. aRNA generated using the optionalIVT Master Mix can be immobilized on a solid substrate displaying NHS-esters via known chemistries. An example of this application can be foundin the following reference:

Wagner P, et al., Bioreactive self-assembled monolayers on hydrogen-passivatedSi(111) as a new class of atomically flat substrates for biological scanning probemicroscopy, J. Struct. Biol., 119(2):189-201 (July 1997).

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RNA Amplification



Application 5: Generation of template for Q-PCR

The aRNA generated using the Paradise Reagent System RNA Amplificationreagents can be used in reactions measuring relative gene expression usingquantitative PCR methods.

The amplification process generates product from the 3’ end of the mRNA.For best results using Q-PCR primer sets should be designed within thefirst 300 bases from the poly (A) tail.

The following protocol may serve as a guide for Q-PCR experiments usingaRNA converted to cDNA as a template.

Protocol

Reverse Transcription

1. Arcturus recommends an aliquot of 100 ng of amplified RNA fromeach sample in a volume of 10 µl.

2. Add 1 µl of 5 mg/ml random hexamer.

3. Mix well by flicking, then briefly spin down by centrifugation for 2minutes in thermal cycler..

4. Incubate samples at 70ºC for 10 minutes. Chill sample to 4ºC.

5. Assemble a master mix with the following components:(components listed are for 1 reaction)

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RT MIXItem Volume (µl) Vendor Catalog #

First Strand Buffer 4 Invitrogen 18064-0220.1Μ DTT 2 Invitrogen 18064-02210 mM dNTP 1 Amersham US77212-500µlRNasin 1 Promega N2511Superscript II, 1 Invitrogen 18064-022Total 9


6. When incubation (Step 4) is complete, mix the tube well by flicking,and then briefly spin down by centrifugation.

7. Add 9 µl of the First Strand Master Mix to each reaction tube.

8. Incubate at 27°C for 10 minutes, followed by 37°C for 1.5 hours inthe thermal cycler.

9. The sample is now ready for Q-PCR.

Q-PCR

Please consult the protocol from the system manufacturer.

RNA Amplification

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B. aRNA Yield and Purity Determination

aRNA quantitation by ultraviolet light absorbance is the simplest approachto determining amplification yield. An absorbance reading at 260 nm(A

260) using a spectrophotometer is taken on a diluted aliquot of aRNA.

Typically, a 1:25 to 1:50 dilution of aRNA in nuclease free water is sufficient.

For single-stranded RNA, a measurement of A260

= 1.0 corresponds to 40µg/mL. The yield can by calculated by:

(A260

) (dilution factor) (40) = µg/mL RNA

Measuring A280

and calculating A260

/A280

ratio indicates the purity of theRNA sample. An A

260/A

280 ratio between 2.0–2.6 indicates very pure

aRNA.

C. Assessment of RNA Sample Quality by QuantitativeReal-Time PCR

Although gel electrophoresis is a common approach to assessing RNAquality, it is not possible to run a gel on the small quantities of input RNAused for amplification with the Paradise Reagent System RNA AmplificationKit. Therefore, you may wish to assess the input RNA quality after 1st

Strand Synthesis using quantitative real-time PCR (Q-RT-PCR). You mayuse the following guidelines:

Use RNase-free technique. Wipe all surfaces and equipment with RNasedecontamination solution, use RNase-free solutions and plasticware, andwear disposable gloves.

1. Perform amplification following the Paradise Reagent System RNAAmplification Kit protocol.

2. During Round One 1st Strand Synthesis, remove 2 µL of the sampleas explained in the optional step #11.

3. Pipette into a new 0.5 mL or 0.2 mL microcentrifuge tube.

4. Add 6 µL 10 ng/µL of Poly I (Sigma, Catalog # P4154). This is thediluted cDNA template.

5. Mix the sample well. Spin down and store on ice until ready to use.

Proceed according to protocols and Instruction Manuals for the Q-RT-PCR system utilized. Use 2 µL of diluted cDNA template (from step 4).Refer to the Q-RT-PCR system manual for details concerning interpretationof data.

The amplification process generates product from the 3’ end of the mRNA.For optimal Q-PCR results, primer sets should be designed within thefirst 300 bases from the poly(A) tail.

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D. Assessment of aRNA Quality Using the Agilent Lab-on-a-Chip System

The Agilent Lab-on-a-Chip system provides a fast and effective approachto assessing the integrity of an aRNA sample. The system requires verysmall quantity of sample. Refer to the Agilent 2100 bioanalyzer and RNALabChip Kit Instruction Manuals for details.

Equipment and Materials RequiredAgilent 2100 bioanalyzer System (Agilent)RNA 6000 Nano Assay Kit (Agilent)Ice or cold block (4°C)Spectrophotometer

Before you begin, refer to the instruction manual for the RNA 6000 NanoAssay Kit. Prepare necessary reagents and supplies as required by the kit.Use RNase-free technique. Wipe all surfaces and equipment with RNasedecontamination solution, use RNase-free solutions and plasticware, andwear disposable gloves.

Protocol1. Determine the concentration of the aRNA generated through Paradise

Reagent System by UV spectrophotometry. (Refer to Appendix B.)

2. Based on the optical density reading, prepare a dilution of the sampleto a concentration of 200 – 300 ng/µL.

3. Store the sample on ice or in a cold block until ready to load on to theRNA chip.

4. Follow the RNA 6000 Nano Assay Kit protocol, loading 1 µL of theprepared sample dilution (from step 2). For details of datainterpretation refer to the bioanalyzer instruction manual. The aRNAappears on the bioanalyzer as a single, broad peak. The size of theaRNA ranges in length from 200 to 2000 bases.

RNA Amplification

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E. Analysis of aRNA by Agarose Gel Electrophoresis

Analysis of aRNA using agarose gel electrophoresis is one method to visualizethe RNA profile and relative quantity after amplification. Standard protocolsfor agarose gel electrophoresis can be used. The following is a suggestedprotocol using commercially available reagents.

Materials1.25% Agarose Portrait Gel or 1.25 Agarose Medium Gel

(EmbiTec cat. # GE-6010 or GE-6030)10X RNA MOPS Running Buffer

(EmbiTec cat. # EC-1020)2X Gel Loading Buffer (various)RNA Ladder (various)SYBR® Gold Nucleic Acid Gel Stain (Molecular Probes cat.# S-11494) or Ethidium Bromide StainNuclease-free Water

Protocol1. Determine the concentration of the aRNA by UV absorbance with a

spectrophotometer. (Refer to Appendix A.)

2. Dilute the aRNA sample(s) with nuclease-free water. Each gel wellcan be loaded with 1 – 3 µg of aRNA.

3. Prepare aRNA gel sample by mixing 6 µL of diluted aRNA with 6 ∝Lof 2X Gel Loading Buffer.

4. Incubate for 3 – 5 minutes at 65°C. Cool on ice.

5. Prepare 1X RNA MOPS Running Buffer and fill gel electrophoresisunit. Place agarose gel into the unit.

6. Load 12 µL of sample per well of the agarose gel. Include RNALadder in one or more lanes.

7. Electrophorese at 5 – 7 volts per centimeter for 30 minutes.

8. Stain the gel with SYBR Gold Nucleic Acid Gel Stain for 30 minutes

or according to the protocol supplied with the reagent. Alternatively,stain with Ethidium Bromide (0.5 – 1.0 µg/mL).

9. Visualize the gel on a UV transilluminator.

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A. Amplification Yield is Poor

1. Cause: Starting RNA sample quality is poor.

Suggestion: If you observe low yields with different RNA samples,run an amplification control using the Control RNA provided in theParadise Reagent System to verify functionality of the kit.

2. Cause: Reagent concentrations in reaction mixtures are incorrect dueto inadequate thawing or mixing.

Suggestion: Ensure all reagents are completely thawed, mixed, and allsolids dissolved prior to use.

3. Cause: Reagent concentrations in the reaction mixtures are incorrectdue to inadequate reaction volume collection in the reaction tube.

Suggestion: Thoroughly thaw and mix all reagents prior to dispensing.Ensure all reagents are dispensed at proper volumes. Briefly spin downthe reaction mix prior to incubation to ensure all reagents are collectedin the reaction volume and the reaction mix has the properconcentrations of reagents.

4. Cause: Reagent concentrations in reaction mixtures are incorrect dueto evaporative condensation onto the wall of the reaction tube duringincubation.

Suggestion: Briefly spin down the sample following incubation stepsto maintain proper volumes and concentrations of reagents and ensurethat all nucleic acid templates are mixed with reaction components.Use a thermocycler with a heated lid.

5. Cause: Incubation temperatures are incorrect.

Suggestion: Verify the accuracy of all incubation temperatures. If youare using a thermal cycler, make sure that the programmed temperaturesread correctly and the instrument has been calibrated to establish andmaintain accurate temperature settings.

6. Cause: RNA yield is diminished during column purification.

Suggestion: Verify centrifugal force used during nucleic acidpurification. Improper binding, washing, and elution centrifugal forcescan decrease the recovery of nucleic acid from the purification column.Microcentrifuges should be calibrated to deliver the correct centrifugalforce.

7. Cause: Message content is low within the total RNA being used inyour study.

Suggestion: Check amplification efficiency using control RNA. Usehigher RNA inputs to compensate for lower message content.

VI. Troubleshooting

RNA Amplification

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B. Low Molecular Weight Product Appears on a Gel

Occasionally, a predominant band below the expected aRNA smear willappear on a gel. This band will lead to improper estimation of yield andmay result in high backgrounds on microarrays. The Paradise ReagentSystem RNA Amplification Kit components are formulated and tested toavoid the synthesis of this material. However, if low molecular weightmaterial is present, one of the following may be occurring:

1. Cause: Quality of the starting RNA is inadequate.

Suggestion: Poor RNA quality can lead to the formation of thereaction artifact, visible as a low molecular weight band. Check thequality of your input RNA. One approach is to utilize the AgilentLab-on-a-Chip System with an RNA LabChip Kit. For additionalrecommendations to check for the quality of the input RNA, contactArcturus Technical Support.

2. Cause: Concentrations of Primer 1, Primer 2, Primer 3, or 1st StrandNuclease Mix are incorrect due to inadequate thawing or dispensing.

Suggestion: Thaw and thoroughly mix each reagent vial prior todispensing. If incompletely thawed and mixed, the concentrations ofthese reagents may not be dispensed at optimal concentrations forthe reaction. Ensure that all pipettes are properly calibrated to dispensecorrect volumes.

3. Cause: Concentrations of Primer 1, Primer 2, Primer 3, or 1st StrandNuclease Mix are incorrect due to inadequate mixing or reactionvolume collection inside the reaction tube.

Suggestion: Thoroughly mix and spin down the sample after addingthe primers or 1st Strand Nuclease Mix into the reaction mix andprior to incubation. This ensures the correct concentration of primersor nuclease in each respective reaction mix.

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