Pronto! (TM) Plus Systems Technical Manual, TM243...I. Description Pronto!™ Plus System(a),...

I. Description ............................................................................................................2

II. Product Components ...........................................................................................4

III. Printing ..................................................................................................................6A. General Considerations...................................................................................6B. Oligonucleotide Arrays.....................................................................................7C. Double-Stranded DNA Arrays..........................................................................8

IV. Total RNA Isolation and Purification...................................................................8A. General Considerations...................................................................................8B. Preparation of Solutions ................................................................................10C. Scraping Procedure for Harvesting and Lysis of Cultured Cells....................11D. Trypsinization Procedure for Harvesting and Lysis of Adherent Cells ...........11E. Harvesting and Lysis of Suspension Cells ....................................................12F. Preparing Lysates from Small Tissue Samples .............................................12G. Purifying RNA by Centrifugation....................................................................14H. Determining RNA Yield and Quality...............................................................15

V. cDNA Labeling and Clean-Up Protocols ..........................................................16A. cDNA Labeling Protocol for Total RNA ..........................................................16B. cDNA Labeling Protocol for mRNA................................................................17C. Purifying Cy®-Labeled cDNA.........................................................................18

VI. Hybridization Protocol .......................................................................................19A. Preparation of Wash Solutions ......................................................................19B. Pre-Soak and Pre-Hybridization ....................................................................20C. Hybridization..................................................................................................21D. Post-Hybridization Washes............................................................................22

VII. Troubleshooting ..................................................................................................23A. Troubleshooting (Printing)..............................................................................23B. Troubleshooting (RNA Isolation) ....................................................................25C. Troubleshooting (cDNA Labeling and Clean-Up)...........................................27D. Troubleshooting (Hybridization) .....................................................................30

VIII. Appendix .............................................................................................................32A. Composition of Buffers and Solutions ...........................................................32B. Average Expected Yields for RNA Isolations .................................................33C. References ....................................................................................................33D. Related Products, Promega Corporation.......................................................34E. Related Products, Corning Incorporated, Life Sciences................................34

Pronto!™ Plus SystemsINSTRUCTIONS FOR USE OF PRODUCTS 40051, 40052, 40053, 40054, 40055, AND 40056.

PLEASE DISCARD PREVIOUS VERSIONS.All technical literature is available on the Internet at www.prontosystems.com

Please visit the web site to verify that you are using the most current version of this Technical Manual.

Technical Manual No. 243

Promega Corporation Toll Free in the USA 800-356-9526 • Tel. 608-274-4330 • Fax 608-277-2516 • [email protected] Incorporated Life Sciences Toll Free in the USA 800-492-1110 • Outside of USA 978-635-2200 • [email protected]

Printed in USA Part# TM243Revised 10/03

I. Description

Pronto!™ Plus System(a), developed jointly by Corning Incorporated and PromegaCorporation, is a fully integrated system of qualified microarray components that pro-vides inter- and intra-lab consistency and reproducibility when generating microarraydata. Microarray users will enjoy the convenience of purchasing all critical microarraycomponents from a single source. All reagents are specifically optimized for micro-array applications using Corning® UltraGAPS™ slides to enable researchers toachieve the highest possible flexibility, performance, standardization, and experi-mental control throughout their microarray processes.

The quality and consistency of the reagents and protocols used to print and processthe microarrays influences the quality and reliability of the microarray results. ThePronto!™ Plus Systems offer reagents and tested protocols to ensure premiumresults. These systems come with a certificate of analysis highlighting functional per-formance and a guarantee of quality and results.

Pronto!™ Plus Systems 1, 3 and 5 include the SV Total RNA Isolation System(b,c),which provides a fast and simple technique for preparing purified and intact totalRNA from a variety of sources in as little as one hour. The isolation procedureincludes a DNase treatment step performed directly on the membrane of the columnto reduce genomic DNA contamination. Purification is achieved without phenol:chloroform extractions or ethanol precipitations, and there is no detectable DNasecarryover.

The ChipShot™ Labeling and ChipShot™ Labeling Clean-Up Systems(d) generatefluorescent cDNAs by direct labeling using Cy®3 and Cy®5 dNTPs. Either total RNAor poly(A)+ mRNA can be used with these systems. Resulting cDNAs are labeled toprovide efficient hybridization results. The ChipShot™ Labeling System includes ahuman Total RNA Positive Control to evalulate the performance of the labeling andlabeling clean-up procedures, helping standardize the microarray experiments. TheChipShot™ Labeling Clean-Up System produces highly purified, labeled cDNAs thatare essentially free of unincorporated nucleotides. Amplification of RNA is notrequired.

Pronto!™ Plus Systems 1 and 2 include the Pronto!™ Microarray Reagent Systemfor printing and hybridizing long oligonucleotide and cDNA microarrays. The printingsubstrate is a major factor that affects microarray quality. Arrays printed on coatedglass of poor quality are likely to produce spots of varying size, shape and DNA con-tent, leading to a loss in sensitivity and generally poor results. UltraGAPS™ slidesoffer a printing surface of unmatched cleanliness, high DNA-binding capacity, unifor-mity and chemical stability.

The Pronto!™ Hybridization Solutions are optimized to create the most favorableenvironment for hybridization between labeled cDNA and spotted amplicons or longoligonucleotides, while minimizing cross-hybridization. Using a starting amount of5µg of total RNA users can detect as little as 3pg target RNA. The Pronto!™Universal Hybridization and Wash Solutions combine to deliver a high level of repro-ducibility, with interslide CVs of 10% or less. The Wash Solutions are formulated toreduce background signal.

The Pronto!™ Plus System provides the reagents and protocols for producing high-quality microarrays and consistent results in one convenient system.


TM243 Printed in USARevised 10/03

Note: For information onmicroarray design consultSambrook and Russell(2001) Molecular Cloning:A Laboratory Manual, 3rd.ed. Cold Spring HarborLaboratory PressA10.1–A10.19 or Ausubel, et al. (1998)Current Protocols inMolecular Biology, Volume3. John Wiley and SonsChapter 22 or reference 1in Section VIII.C.



RNA Isolation

~ 1 hour Isolate Total RNA using the SV Total RNA Isolation System (poly (A)+RNA should be isolated according to vendor recommendations).

3 hours Prepare cyanine dye-labeled cDNA by reverse transcription of total RNA or poly (A)+ RNA.

0.75 hour Purify cyanine dye-labeled cDNA.

0.5 hour Quantitate cDNA by spectrophotometry.

~ 1.5 hours Pre-Soak DNA microarrays and prepare for hybridization.

Overnight Hybridize DNA at 42°C.

1 hour Perform stringent washes.

0.25 hour Scan slide using fluorescent microarray scanner.

Printing

cDNA Synthesis

4250

MA

07_3

A

Flow Diagram Comments

cDNA Purification

cDNA Quantitation

Pre-Soak/Pre-Hybridization

Hybridization

Post-HybridizationWashes

Scanning

Printing time is variable depending on array density and post processing.



II. Product Components

Product Size Cat.#Pronto!™ Plus System 1 10 reactions 40051This system contains sufficient reagents to perform 10 total RNA preparations, label andpurify 10 cDNAs plus one control, and prepare and process 10 microarrays. Includes:

• 1 Pronto!™ Universal Microarray Validation Kit (10 reactions; includes UltraGAPS™Slides, Spotting Solution and Pre-Soak Tablets)

• 1 SV Total RNA Isolation System (10 preparations)• 1 ChipShot™ Labeling System (10 experimental reactions plus 1 control)• 1 ChipShot™ Labeling Clean-Up System (11 reactions)• 1 Protocol

Product Size Cat.#Pronto!™ Plus System 2 10 reactions 40052This system contains sufficient reagents to label and purify 10 cDNAs plus one control andprepare and process 10 microarrays. Includes:

• 1 Pronto!™ Universal Microarray Validation Kit (10 reactions, includes UltraGAPS™Slides, Spotting Solution and Pre-Soak Tablets)

• 1 ChipShot™ Labeling System (10 experimental reactions plus 1 control)• 1 ChipShot™ Labeling Clean-Up System (11 reactions)• 1 Protocol

Product Size Cat.#Pronto!™ Plus System 3 10 reactions 40053This system contains sufficient reagents to perform 10 total RNA preparations, label andpurify 10 cDNAs plus one control and process 10 microarrays. Includes:

• 1 Pronto!™ Universal Microarray Hybridization Kit (10 reactions, DOES NOTinclude UltraGAPS™ Slides or Spotting Solution)

• 1 SV Total RNA Isolation System (10 preparations)• 1 ChipShot™ Labeling System (10 experimental reactions plus 1 control)• 1 ChipShot™ Labeling Clean-Up System (11 reactions)• 1 Protocol

Product Size Cat.#Pronto!™ Plus System 4 10 reactions 40054This system contains sufficient reagents to label and purify 10 cDNAs plus one control andprocess 10 microarrays. Includes:

• 1 Pronto!™ Universal Microarray Hybridization Kit (10 reactions; DOES NOTinclude UltraGAPS™ Slides or Spotting Solution)

• 1 ChipShot™ Labeling System (10 experimental reactions plus 1 control)• 1 ChipShot™ Labeling Clean-Up System (11 reactions)• 1 Protocol

Product Size Cat.#Pronto!™ Plus System 5 25 reactions 40055This system contains sufficient reagents to perform 25 total RNA preparations, label and purify25 cDNAs (24 experimental and 1 control) and process 25 microarrays. Includes:

• 1 Pronto!™ Universal Microarray Hybridization Kit (25 reactions, DOES NOTinclude UltraGAPS™ Slides)

• 1 SV Total RNA Isolation System (25 preparations)• 1 ChipShot™ Labeling System (25 reactions)• 1 ChipShot™ Labeling Clean-Up System (25 reactions)• 1 Protocol



Product Size Cat.#Pronto!™ Plus System 6 25 reactions 40056This system contains suffient reagents to label and purify 25 cDNAs (24 experimental and 1 control) and process 25 microarray hybridizations. Includes:

• 1 Pronto!™ Universal Microarray Hybridization Kit (25 reactions; DOES NOTinclude UltraGAPS™ Slides)

• 1 ChipShot™ Labeling System (25 reactions)• 1 ChipShot™ Labeling Clean-Up System (25 reactions)• 1 Protocol

Storage Conditions: All of the components of the Pronto!™ Universal MicroarrayValidation Kit and the Pronto!™ Universal Microarray Hybridization Kit can be storedat normal laboratory ambient temperatures (20–27°C). All kit components have metfunctional performance criteria after exposure to temperatures between –20°C and45°C.

Store UltraGAPS™ slides at ambient temperature (20–27°C) in original, undam-aged packaging, and use slides by the date indicated on the label. Proceed asdescribed in the Handling and Care Instructions, found in Section III, after openingthe package.

Store the SV Total RNA Isolation System and the ChipShot™ Labeling Clean-UpSystem at room temperature. Store the ChipShot™ Labeling System at –20°Cexcept for the RNase A, which should be stored at room temperature, and the TotalRNA Positive Control, which should be stored at –70°C.

Safety Considerations: When working with the Pronto!™ Plus System please follow all generally accepted laboratory safety guidelines. At a minimum, wear appro-priate personal protective equipment such as a lab coat, safety glasses, powder-freelatex gloves, etc. Follow recommended standard operating procedures for any labo-ratory equipment used in your experiments. Read all Material Safety Data Sheets forappropriate handling of all reagents provided in the Pronto!™ Plus System. MSDSare available upon request or can be downloaded from the Promega web site at:www.prontosystems.com

Product Use Limitations, Warranty, Disclaimer

Pronto!™ Plus Systems are sold for research purposes only and are not intended forresale. This product is not to be used in human diagnostics or for drug purposes, noris it to be administered to humans in any way. This product contains chemicals thatmay be harmful if misused. Proper care should be exercised with this product to pre-vent human contact. These products are guaranteed to perform as described whenused properly. Manufacturer liability is limited to the replacement of the product or afull refund. Any misuse of this product including failure to follow proper use protocolsis the responsibility of the user, and Corning and Promega make no warranty orguarantee under these circumstances.

Certain arrays and/or methods of preparation, analysis or use may be covered byintellectual property rights held by others in certain countries. Use of this product isrecommended only for applications for which the user has a license under propri-etary rights of third parties or for technology for which a license is not required.

Store the ...Total RNA

Positive Control at –70°Cfor long-term storage.

!

III. Printing

A. General Considerations

The UltraGAPS™ slide surface is highly reactive toward DNA. The key to pro-ducing high-quality microarrays is to take advantage of this high reactivity duringthe printing process while minimizing the spurious attachment of nucleic acids tothe unprinted area during subsequent manipulation of the array. Critical factorsto consider include:

Concentration of the DNA. The high reactivity of UltraGAPS™ slides allowsthe use of dilute printing solutions. The optimal concentration needs to be deter-mined empirically. When too little DNA is used, the printed spots will not reachsignal saturation levels, thus reducing the dynamic range of the array.Conversely, highly concentrated printing solutions can produce spots with“comet tails” and other forms of localized background. The concentration andpurity of the DNA should be checked spectrophotometrically as well as elec-trophoretically. We recommend 0.1mg/ml as a starting point for further optimiza-tion when printing dsDNA (e.g., PCR products, genomic DNA) and 0.5mg/mlwhen printing oligonucleotides.

Size of Oligonucleotides. UltraGAPS™ slides are optimized for use with longoligonucleotides. Oligos greater than 50 nucleotides (nts) in length (optimally75nts) demonstrate superior hybridization efficiency and specificity on theUltraGAPS™ surface.

Arrayer Settings and Pin Quality. Follow the instructions provided by the manufacturer of arraying equipment and printing pins. Pin-contact time and theforce with which the pin strikes the slide affect spot size and morphology. Pinsmust be individually qualified before use. Pins that are either broken or do notconform to specifications can ruin otherwise good arrays. Make sure to optimizethe printing and pin-washing steps before using the Pronto!™ Universal SpottingSolution for the first time.

Immobilization Procedures. UV crosslinking and/or baking enhances bindingof DNA to the GAPS-coated surface. These procedures work equally well forDNA molecules longer than 300bp. Smaller DNA molecules and oligo-nucleotides are best immobilized by UV crosslinking. When baking, care shouldbe taken regarding the cleanliness of the oven. Volatile organics can irreversiblycontaminate the surface of the array, leading to high backgrounds.

Procedures To Reduce Background Levels. Depending on their age, thepurity of the biological material and other reagents used, and the storage condi-tions, DNA microarrays may develop significant levels of background fluores-cence on and around the printed areas. It is important to eliminate “spotted” fluo-rescence in order to accurately measure basal levels of transcript abundance.The Pronto!™ Universal Pre-Soak treatment followed by Pre-Hybridization, asdetailed in Section VI, is efficient at eliminating background fluorescence.

Handling and Care of UltraGAPS™ Slides

To maximize the benefits of using Pronto!™ premium substrates and reagents,please follow these recommendations:

1. Use the slides in a clean environment. Particles falling onto the slide sur-face may cause defects in the printed array as well as nuclease contamina-tion. Self-contained printing environments may be required to prevent suchcontamination.



For technical assistancewith microarray printing,please contact CorningIncorporated, Life Sciences. Contactinformation is available at:www.corning.com/lifesciences

E-mail:[email protected]

Note: For information onmicroarray design consultSambrook and Russell(2001) Molecular Cloning:A Laboratory Manual, 3rd.ed. Cold Spring HarborLaboratory PressA10.1–A10.19 or Ausubel, et al. (1998)Current Protocols inMolecular Biology, Volume3. John Wiley and Sons,Chapter 22 or reference 1in Section VIII.C.

2. Avoid direct contact with the surface of the slide to be printed. Only the printpins and processing solutions should touch the print area to avoid contami-nation and abrasion of the coating.

3. When using slides without bar codes, always print on the side facing awayfrom the wall of the plastic container. Clearly mark the side to be printedusing a glass-etching tool.

4. If the package of slides has been inadvertently stored at temperatures lowerthan 20°C, allow it to come to room temperature before opening. Otherwise,condensation may form on the slide surface, negatively affecting the unifor-mity of the coating.

5. Open the pouch just prior to printing. Close the cap on the slide container assoon as possible after removing slides for use to maintain a closed environ-ment for unused slides. Place the closed container in the pouch to protectthe remaining slides and store them in a desiccator. Use the remainingslides within one week of opening the pack.

Materials to Be Supplied by the User

• arrayer• DNA source plates (e.g., Corning Cat.# 3656)• vacuum desiccator• UV light source• oligonucleotides or double-stranded DNA

Materials Supplied in the Following Systems: 40051 and 40052

• Pronto!™ Universal Spotting Solution• UltraGAPS™ Slides

The Pronto!™ Universal Spotting Solution is provided ready for use. Dilution oraddition of other reagents is not necessary. The Pronto!™ Universal SpottingSolution is an excellent medium for dissolving oligonucleotides as well as dsDNAfor printing microarrays. This proprietary formulation has been tested thoroughlyon UltraGAPS™ slides and may be used with either solid or quill pins.

B. Oligonucleotide Arrays

1. Prepare DNA source plates (sterile, nuclease-free Corning® 384 WellStorage Microplates are recommended; Cat.# 3656) by one of either alternative methods a or b. Sufficient volume of Pronto!™ Universal SpottingSolution needs to be prepared to cover the bottom of the receiving wells;this corresponds to between 5 and 10µl per well when using 384-well plates.Please follow the recommendations of the microarrayer manufacturer.

a. Dissolve oligonucleotides to a maximum of 1.0mg/ml (0.5mg/ml is agood starting concentration for further optimization) in Pronto!™ UniversalSpotting Solution. Transfer DNA solution to the Corning® 384-well plate.

b. Alternatively, add the desired volume of Pronto!™ Universal SpottingSolution to wells containing DNA dried by vacuum centrifugation.

2. Set up arrayer and print slides according to the manufacturer’s or laboratoryprotocol. The printing environment should be free of dust particles and keptat a temperature of 20–22°C with relative humidity between 45 and 55%.

3. Place arrays in a desiccator for up to 48 hours (vacuum desiccator works best).



Note: To approximatebetween molarity and mass use the followingequations.

XµM = (3000 × Ymg/ml)N

Ymg/ml = (XµM × N)3000

where N = number ofnucleotides

4. Immobilize spotted oligonucleotides by applying 600mJ of UV energy to theprinted surface.

5. Store arrays in a dry environment at normal laboratory temperature (20–25°C).The orange-colored plastic containers may be used for long-term storage ofthe arrays. Arrays can be stored for up to 6 months prior to hybridization.Exchanging the regular atmospheric air with clean nitrogen gas helps preventoxidation of spotted material and extends the shelf life of the arrays.

C. Double-Stranded DNA Arrays

1. Prepare DNA source plates (sterile, nuclease-free Corning® 384 WellStorage Microplates are recommended; Cat.# 3656) by one of either alter-native methods a or b. Sufficient volume of Pronto!™ Universal SpottingSolution needs to be prepared to cover the bottom of the receiving wells;this corresponds to between 5 and 10µl per well when using 384-well plates.Please follow the recommendations of the microarrayer manufacturer.

a. Dissolve dsDNA to a maximum of 0.25mg/ml (0.1mg/ml is a good start-ing concentration for further optimization) in Pronto!™ Universal SpottingSolution. Transfer DNA solution to the Corning® 384-well plate.

b. Alternatively, add the desired volume of Pronto!™ Universal SpottingSolution to wells containing DNA that has been dried by vacuum centrifugation.

2. Set up arrayer and print slides according to the manufacturer’s or laboratoryprotocol. The printing environment should be free of dust particles and keptat a temperature of 20–22°C with relative humidity between 45 and 55%.

3. Place arrays in a desiccator for up to 48 hours (vacuum desiccator works best).

4. Immobilize spotted DNA by applying 150 to 300mJ of UV light to the printedsurface of the array or by baking the array at 80°C for 2–4 hours.

5. Store arrays in a dry environment at normal laboratory temperature (20–25°C).The orange-colored plastic containers may be used for long-term storage ofthe arrays. Arrays can be stored for up to 6 months prior to hybridization.Exchanging the regular atmospheric air with clean nitrogen gas helps preventoxidation of spotted material and extends the shelf life of the arrays.

IV. Total RNA Isolation and Purification

Pronto!™ Plus Systems 40051, 40053, and 40055 contain the reagents necessaryto purify total RNA. Sections IV.C, D and E of this manual provide protocols for har-vesting and lysis of cultured cells. Section IV.F provides the protocol for lysis andhomogenization of tissue samples ≤30mg.

To isolate total RNA from white blood cells, plant tissue, yeast and bacterial cellsor from tissue quantities >30mg, refer to the SV Total RNA Isolation System TechnicalManual, #TM048, which can be found at: www.promega.com/tbs/tm048/tm048.html

A. General Considerations

For best results from this system when processing tissue, use fresh samples.Older samples may yield less total RNA. If necessary, freeze the samples immedi-ately after collection in liquid nitrogen and store at –70°C for future use. Sampleshomogenized in SV RNA Lysis Buffer may be stored at –20 or –70°C. For valuabletissue samples, we suggest that a portion of each sample be reserved at –70°C inthe event that loss of a sample occurs during RNA purification.



For technical assistancewith RNA isolation andpurification, please con-tact your local PromegaBranch Office or distributor. Contactinformation available at:www.promega.com


Processing Capacity. The SV Total RNA Isolation System is developed andoptimized for total RNA isolations from tissues or cultured cells with a broadspectrum of RNA expression levels. The maximum processing capabilities of asingle SV RNA column are 2 x 106 cultured cells or 30mg tissue in 175µl LysisBuffer. Certain tissues may be processed using more than 30mg/175µl, and recommendations for these tissues are provided in Table 1, Section IV.F. Excesstissue should not be processed because this clogs the membranes. The maxi-mum volume of lysate that can be processed in each Spin Basket is 175µl.

Creating a Ribonuclease-Free Environment. Ribonucleases are extremelydifficult to inactivate. Care should be taken to avoid inadvertently introducingRNase activity into your RNA during or after the isolation procedure. This isespecially important if the starting material has been difficult to obtain or is irreplaceable. The following notes may help you to prevent accidental RNasecontamination of your sample.

1. Two of the most common sources of RNase contamination are the user'shands and bacteria or molds present on airborne dust particles. To preventcontamination from these sources, use sterile technique when handling thereagents supplied with the system. Gloves should be worn at all times.

2. Whenever possible, sterile disposable plasticware should be used for han-dling RNA. These materials are generally RNase-free and thus do notrequire pretreatment to inactivate RNase. RNase-free Elution Tubes are provided with the system.

3. Treat nondisposable glassware and plasticware before use to ensure that itis RNase-free. Bake glassware at 200°C overnight, and thoroughly rinseplasticware with 0.1N NaOH, 1mM EDTA followed by RNase-free water.

4. Treat solutions supplied by the user by adding diethyl pyrocarbonate(DEPC) to 0.1% and then incubating overnight at room temperature.Autoclave for 30 minutes to remove any trace of DEPC.

For downstream applications, protect your RNA samples from RNases bywearing gloves and using solutions and centrifuge tubes that are RNase-free.

Materials to be Supplied by the User(Solution compositions are provided in Section VIII.A)

Specific supplies for use with tissue samples

• small tissue homogenizer (e.g., Tekmar Tissuemizer™)• scale, sterile razor blade• liquid nitrogen• mortar and pestle



DO NOT...treat Tris buffers withDEPC. DEPC reactsrapidly with amines andcannot be used to treatTris buffers.

• gloves• RNase-free pipettes• 1X trypsin-EDTA solution

(for adherent cells)• 1X PBS (for cultured cells)• cell growth media• 37°C incubator• sterile centrifuge tube• centrifuge capable of 300 × g

• microcentrifuge capable of12,000 × g

• microcentrifuge tube rack• microcentrifuge tubes• sterile hypodermic syringe fitted

with a sterile 20-gauge needle • water bath or heating block, pre-

heated to 70°C• ethanol, 95%, RNase-free

CAUTION: DEPC is a suspected carcinogen andshould be used in a chemical fume hood.

Materials Supplied in the Following Systems: 40051, 40053 and 40055

B. Preparation of Solutions

Before beginning the SV Total RNA Isolation System protocol, four solutionsmust be prepared.

Solution Preparation Steps NotesSV RNA Lysis Buffer 25 prep size (Cat.# Z3102): After adding BME,

Add 1ml β-Mercaptoethanol mark on the bottle that (BME) to 50ml of SV RNA you have performed this Lysis Buffer. step. Store the SV RNA

Lysis Buffer at 4°C.10 prep size (Cat.# Z3101):Add 200µl β-Mercaptoethanol (BME) to 10ml of SV RNA Lysis Buffer.

DNase I 25 prep size (Cat.# Z3102): We recommend dividing Add the amount of Nuclease- the rehydrated DNase I Free Water (supplied) indicated into working aliquotson the DNase I vial to the (e.g., 5–10 equal aliquots)lyophilized DNase I. using sterile RNase-free

microcentrifuge tubes.10 prep size (Cat.# Z3101): A total of 5µl ofAdd the amount of Nuclease- rehydrated DNase I isFree Water (supplied) indicated required per RNA on the DNase I vial to the purification. Store rehy-lyophilized DNase I. drated DNase I at –20°C.Gently mix by swirling the vial.Do not vortex.

SV RNA Wash Solution 25 prep size (Cat.# Z3102): After adding ethanol,Add 100ml of 95% ethanol to mark on the bottle labelthe bottle containing 58.8ml that you have performedconcentrated SV RNA this step. The reagent isWash Solution. stable at 22–25°C when

tightly capped.10 prep size (Cat.# Z3101):Add 20ml of 95% ethanol to the bottle containing 11.8mlconcentrated SV RNA Wash Solution.

SV DNase Stop Solution 25 or 10 prep size (Cat.# Z3102 After adding ethanol,or Cat.# Z3101): Add 8ml 95% mark on the bottle labelethanol to the bottle containing that you have performed5.3ml concentrated SV DNase this step. The reagent is Stop Solution. stable at 22–25°C when

tightly capped.



For technical assistancewith RNA isolation andpurification, please con-tact your local PromegaBranch Office or distributor. Contactinformation available at:www.promega.com


DO NOT...vortex the DNase I solution.

DO NOT...freeze-thaw aliquots ofrehydrated DNase I.

• Spin Column Assemblies andElution Tubes (5 each/pack)

• SV RNA Lysis Buffer• SV RNA Dilution Buffer (blue

buffer)• β-mercaptoethanol (48.7%)• DNase I (lyophilized)

• MnCl2, 0.09M• Yellow Core Buffer• SV DNase Stop Solution

(concentrated)• SV RNA Wash Solution

(concentrated)• Nuclease-Free Water

C. Scraping Procedure for Harvesting and Lysis of Cultured Cells

Use at least 1.5 x 103 cells to a maximum of 2 x 106 cells per purification. Thenumber of cells used may need to be adjusted depending on cell type, functionand RNA expression levels at the time of harvest. For adherent cells grown inmultiwell plates, a yield of 3.5 x 104–1 x 106 cells per well is possible dependingon the cell type and well size.

1. Lysates may be prepared rapidly by manually scraping the cells from theculture vessel in the presence of SV RNA Lysis Buffer. Recommended vol-umes of SV RNA Lysis Buffer per well are 175µl for 6- to 96-well plates anda minimal volume (e.g., 1–2ml) for a flask.

2. Add SV RNA Lysis Buffer (with ethanol added), scrape and pipet to mix.

Note: For 6- to 12-well plates, pipet the SV RNA Lysis Buffer over the bot-tom of the well to contact all of the cells. Scraping is not required for individ-ual wells.

3. Transfer 175µl of the cell lysate to a sterile 1.5ml screw cap or microcen-trifuge tube.

4. Add 350µl of SV RNA Dilution Buffer to 175µl of cell lysate. Mix by invertingthe tube 3–4 times.

5. Place in a water bath or heating block at 70°C for 3 minutes. Incubatinglonger than 3 minutes may compromise the integrity of the RNA.

6. Centrifuge at 12,000–14,000 × g for 10 minutes at 20–25°C. Proceed toSection IV.G (Purifying RNA by Centrifugation). To obtain a protocol for puri-fying RNA by vacuum, please see the SV Total RNA Isolation SystemTechnical Manual #TM048 (www.promega.com/tbs/tm048/tm048.html).

D. Trypsinization Procedure for Harvesting and Lysis of Adherent Cells

Use at least 1.5 x 103 cells to a maximum of 2 x 106 cells per purification. Thenumber of cells used may need to be adjusted depending on cell type, functionand RNA expression levels at the time of harvest.

1. Remove the media and wash the cells with sterile 1X PBS.

2. Prepare a sterile trypsin-EDTA solution in 1X PBS. Remove the 1X PBS washfrom the cells, and add just enough of the trypsin-EDTA solution to cover thecell monolayer: 2ml for a 150mm flask, 1ml for a 100mm plate. Rock the cul-ture vessel to distribute the trypsin solution evenly. Place the plates or flask in a37°C incubator until the cells just begin to detach (usually 1–2 minutes).

3. Add cell growth media (up to 50ml) to the plates or flasks, and mix to detachthe cells from the surface.

4. Transfer the media and cells to a sterile 50ml conical centrifuge tube, andpellet cells by centrifugation at 300 × g for 5 minutes. If cells remain in thesupernatent after centrifugation, repeat the centrifugation at 300 × g for 5additional minutes. Discard the supernatant.

5. Wash the cell pellet by resuspending in 50ml of sterile 1X PBS. Centrifugeat 300 × g for 5 minutes to collect the cells. Discard the supernatant.

6. Verify that BME has been added to the SV RNA Lysis Buffer. Add 175µl ofSV RNA Lysis Buffer to the washed cells, dispersing the pellet and mixingwell by vortexing and/or pipetting.



Note: To isolate totalRNA from white bloodcells, plant tissue, yeastand bacterial cells orfrom tissue quantities>30mg, refer to the SVTotal RNA IsolationSystem TechnicalManual, #TM048, whichcan be found at:www.promega.com/tbs/tm048/tm048.html

7. Transfer the 175µl of cell lysate to a sterile 1.5ml screw cap or microcen-trifuge tube.

8. Add 350µl of SV RNA Dilution Buffer to 175µl of lysate. Mix by inverting thetube 3–4 times.


10. Centrifuge at 12,000–14,000 × g for 10 minutes at 20–25°C. Proceed toSection IV.G. (Purifying RNA by Centrifugation). To obtain a protocol for puri-fying RNA by vacuum, please see the SV Total RNA Isolation SystemTechnical Manual #TM048 (www.promega.com/tbs/tm048/tm048.html).

E. Havesting and Lysis of Suspension Cells

1. Collect 1.5 x 103–2 x 106 cells in a sterile 50ml conical centrifuge tube bycentrifugation at 300 × g for 5 minutes.

2. Wash the cell pellet by resuspending in 25ml of sterile 1X PBS. Centrifugeat 300 × g for 5 minutes to collect the cells. Discard the supernatant.

3. Verify that BME has been added to the SV RNA Lysis Buffer. Add 175µl ofSV RNA Lysis Buffer to the washed cells, dispersing the pellet and mixingwell by vortexing and/or pipetting.

4. Transfer the 175µl of cell lysate to a sterile 1.5ml screw cap or microcen-trifuge tube.

5. Add 350µl of SV RNA Dilution Buffer to 175µl of lysate. Mix by inverting thetube 3–4 times.


7. Centrifuge at 12,000–14,000 × g for 10 minutes at 20–25°C. Proceed toSection IV.G (Purifying RNA by Centrifugation). To obtain a protocol for puri-fying RNA by vacuum, please see the SV Total RNA Isolation SystemTechnical Manual #TM048 (www.promega.com/tbs/tm048/tm048.html).

F. Preparing Lysates from Small Tissue Samples

This protocol is for processing small tissue samples. In general, the mass of tissue to be lysed and homogenized in 175µl SV RNA Lysis Buffer should be≤≤30mg. To purify RNA from larger sample sizes, multiple isolations will be nec-essary. Consider the 50 prep size (Promega Cat.# Z3100).

1. Transfer 175µl of SV RNA Lysis Buffer (with BME added) to a sterile micro-centrifuge tube to which your tissue sample will be added. Use RNase-freepipettes, and wear gloves to reduce the chance of RNase contamination.

2. Weigh the tube containing the SV RNA Lysis Buffer and record the weight.

3. Quickly cut the tissue into small pieces with a sterile razor blade, freeze in liquid nitrogen, and grind in a mortar and pestle under liquid nitrogen. Transferliquid nitrogen and ground tissue to an appropriately sized sterile tube, allowthe liquid nitrogen to evaporate and then immediately transfer the tissue to thetube containing 175µl SV RNA Lysis Buffer. Mix thoroughly by inversion.



Note: The maximumamount of lysate that canbe efficiently processed is175µl per spin basket.

4. Weigh the tube containing the tissue and SV RNA Lysis Buffer. Calculate thetissue mass by subtracting the weight obtained in Step 2 from this new weight.In general, the ratio of tissue mass to SV RNA Lysis Buffer should be approxi-mately 30mg/175µl but may be adjusted for certain tissues (see Table 1 for rec-ommendations for tissue mass to SV RNA Lysis Buffer ratios for different tis-sues). If necessary, add SV RNA Lysis Buffer to the tissue to achieve this ratio.

Note: Some lysed samples (e.g., spleen) contain a large amount of nucleicacid, cellular debris and protein, which cause a very thick lysate. If the sampleis too viscous to pipet easily, dilute with additional SV RNA Lysis Buffer beforeadding the SV RNA Dilution Buffer in Step 5.The maximum volume of lysatethat can be processed in each Spin Basket is 175µl. For tissues with lowerRNA levels, more concentrated lysate may be used as long as the lysate is notdifficult to pipet.

5. Add 350µl of SV RNA Dilution Buffer to 175µl of lysate. Mix by inverting 3–4times. Place in a water bath or heating block at 70°C for 3 minutes.Incubating longer than 3 minutes may compromise the integrity of the RNA.

6. Centrifuge for 10 minutes at 12,000–14,000 × g. Proceed to Section IV.G(Purifying RNA by Centrifugation). To obtain a protocol for purifying RNA byvacuum, see the SV Total RNA Isolation System Technical Manual #TM048(www.promega.com/tbs/tm048/tm048.html).



Note: If the lysate is tooviscous to pipet easily,dilute with additional SVRNA Lysis Buffer beforeadding SV RNA DilutionBuffer. Add the minimumamount of SV RNA LysisBuffer that is required tomake the lysate easy topipet.

If this is done, the amountof dilution buffer needs tobe increased so that thesame ratio of lysis buffer todilution buffer is retained.

Table 1. Recommended Amounts of Tissue and Cells for Preparation of Lysates.

Sample Approximate Wet Weight of Maximum Tissue Mass or Number ofMaterial Organ from a 30g Mouse Cells per 175µl Lysis Buffer

Liver 940mg 30mgKidney 210mg 20mgMuscle – 30mgSpleen 90mg 15mgHeart 150mg 60mgBrain 463mg 60mgLung 200mg 60mgRAW264.7 cells not applicable 1 x 105–5 × 106

The tissue amounts and cell numbers for given volumes of SV RNA Lysis Buffer are recom-mended for optimal performance of the Spin Columns.The maximum amount of lysate thatcan be efficiently processed is 175µl per Spin Basket. If a lysate contains more RNA thanthe capacity of the Spin Basket allows, some RNA will be lost during the wash steps. Anapproximate weight for all types of muscle is not given.

Sample Material Protocol

Plant Cells See Technical Manual #TM048 for a White Blood Cells protocol for purifying total RNA from theseYeast sample types using the SV Total RNABacteria Isolation System.

G. Purifying RNA by Centrifugation

Five Spin Column Assemblies (Spin Basket and Collection Tube) and fivecapped Elution Tubes (1.5ml) are packed together. If you do not need the capson the Spin Baskets simply remove them using a twisting motion; the caps aredesigned to detach from the Spin Baskets. If the caps are left attached to theSpin Baskets, they must be closed during centrifugation steps. Label theCollection Tube and place the Spin Basket in the Collection Tube. Wear gloveswhen handling the tubes.

1. Transfer the cleared lysate to a fresh 1.5ml microcentrifuge tube by pipet-ting. Avoid disturbing the pelleted debris.

2. Add 200µl 95% ethanol to the cleared lysate in the fresh microcentrifugetube and mix by pipetting 3–4 times.

3. Transfer this mixture to the Spin Basket in the Collection Tube. Centrifuge at12,000–14,000 × g for 1 minute.

4. Remove and retain the Spin Basket from the Collection Tube and discardthe liquid in the Collection Tube. Return the Spin Basket back to theCollection Tube.

5. Verify that the SV RNA Wash Solution has been diluted with ethanol asdescribed in Section IV.B. Add 600µl of SV RNA Wash Solution to the SpinBasket in the Collection Tube.

6. Centrifuge at 12,000–14,000 × g for 1 minute.

7. Remove and retain the Spin Basket from the Collection Tube and discardthe liquid in the Collection Tube. Return the Spin Basket back to theCollection Tube and place in a rack.

8. For each isolation to be performed, prepare the DNase incubation mix in asterile tube on ice by combining the following components in the order listed:

Yellow Core Buffer 40µl0.09M MnCl2 5µlDNase I enzyme 5µl

Prepare only the amount of DNase incubation mix required. Mix by gentlepipetting; do not vortex.

9. Apply 50µl of this freshly prepared DNase incubation mix directly to themembrane inside the Spin Basket. Make sure that the solution is in contactwith and thoroughly covering the membrane. The incubation solution is yellow to make this easier to visualize.

10. Incubate for 15 minutes at 20–25°C.

11. After this incubation, add 200µl of SV DNase Stop Solution (as prepared inSection IV.B; verify that ethanol has been added) to the Spin Basket.

12. Centrifuge at 12,000–14,000 × g for 1 minute. There is no need to empty theCollection Tube before the next step.

13. Add 600µl SV RNA Wash Solution to the Spin Basket, and centrifuge at12,000–14,000 × g for 1 minute.



DO NOT.. mix the Yellow CoreBuffer and 0.09M MnCl2before Step 8. The YellowCore Buffer and 0.09MMnCl2 should be storedseparately and mixedfresh for each set of RNApreparations.

Carryover...of a small amount ofpelleted debris is not detri-mental to RNA purifica-tion. Sometimes debriswill form a solid layer ontop of the supernatant.Simply push this layer tothe side with the pipettetip before pipetting thesupernatant. The super-natant volume should beapproximately 500µl, butwill depend on the amountof tissue mass in thelysate.

!



14. Remove and retain the Spin Basket from the Collection Tube and discardthe liquid in the Collection Tube. Return the Spin Basket back to theCollection Tube and set in a rack.

15. Add 250µl SV RNA Wash Solution to the Spin Basket and centrifuge at12,000–14,000 × g for 2 minutes.

16. If you have not already done so, remove the cap from the Spin Basket usinga twisting motion. For each sample, remove one capped 1.5ml Elution Tubefrom the packaging. Note that there is only one Elution Tube per SpinBasket. Place the Spin Basket in the Elution Tube.

17. Add 100µl Nuclease-Free Water to the Spin Basket membrane. Be sure tocompletely cover the surface of the membrane with the water. IncubateNuclease-Free Water for 2 minutes at ambient temperature.

18. Place the Elution Tube and Spin Basket in the centrifuge with the lid of theElution Tube facing out. Centrifuge at 12,000–14,000 × g for one minute.

19. Remove the Spin Basket and discard.

20. Cap the Elution Tube containing the purified RNA and store at –70°C.

Note: Elution volumes of less than 100µl are not recommended. If the RNAneeds to be concentrated, it can be precipitated or vacuum-dried and resus-pended in a smaller volume of water. For guidance on precipitating RNA,see the “Low concentration” heading in Section VII.B, Troubleshooting. Ifmaximum recovery of RNA is essential, a second elution into a second ster-ile tube with an additional 100µl of Nuclease-Free Water followed by cen-trifugation at 12,000–14,000 × g for 1 minute is recommended. Dependingon the amount of input tissue and RNA expression levels, a second elutionmay yield as much as 10–20% additional RNA.

H. Determining RNA Yield and Quality

The SV Total RNA Isolation System can be used to generate intact RNA from a vari-ety of tissues and cell sources.The yield of total RNA obtained may be determinedspectrophotometrically at 260nm, where 1 absorbance unit (A260) equals 40µg of sin-gle-stranded RNA/ml.The purity may also be estimated by spectrophotometry fromthe relative absorbances at 230, 260 and 280nm (i.e., A260/A280 and A260/A230).

Pure RNA will exhibit an A260/A280 ratio of 2.0. However, it should be noted that,due to the variations between individual starting materials and in performing theprocedure, the expected range of A260/A280 ratios for RNA will be 1.7–2.1. If theRNA exhibits a ratio less than 1.7, refer to Troubleshooting (Section VII.B) for pos-sible causes and suggestions on improving the purity of the RNA. Table 4(Section VIII.B) lists representative yields and A260/A280 ratios of total RNA iso-lated from a number of different cell and tissue sources using the SV Total RNAIsolation System. Using this protocol, the RNA will usually exhibit an A260/A230

ratio of 1.8–2.2. A low A260/A230 ratio may indicate guanidine contamination thatcan interfere with downstream processing.

Special Note: For some sample purifications RNA yield may need to be deter-mined using RiboGreen® RNA Quantitation Reagent Kit (Molecular ProbesCat.# R11490) to conserve precious sample for downstream labeling (3).

Note: Elution volumes ofless than 100µl are not rec-ommended. If the RNAneeds to be concentrated,it can be precipitated orvacuum-dried and resus-pended in a smaller volumeof water.

Note: Refer to SectionVIII.B, Table 4 for expectedRNA yields.

Note: The A260 observedwith most other RNA purifi-cation systems includesthe contribution from con-taiminating DNA. The DNAcontribution results in anoverestimation of the RNAyield. The genomic DNA iseliminated by the DNasetreatment step in the SVTotal RNA Isolation Systemprotocol, resulting in moreaccurate estimation of theRNA yield.

V. cDNA Labeling and Clean-UP Protocols

The quality and cleanliness of the starting RNA and the resulting cDNA are critical factors for successful use of arrays. We recommend that RNA quality bethoroughly checked before attempting to synthesize cDNA and that the labeledcDNA be purified and quantified using a spectrophotometer. Minimize the expo-sure of solutions containing fluorescent nucleotides to light to prevent photo-bleaching. Cy®-labeled cDNA is stable at 4°C for several weeks.

We recommend that you wear gloves throughout the cDNA synthesis procedure.

For information on mRNA isolation, please see the PolyATtract ® mRNA IsolationSystems (Promega Cat.# Z5200 and Z5300) Technical Manual #TM021(www.promega.com/tbs/tm021/tm021.html). mRNA prepared using this systemhas been used with microarrays (see references 4–8 Section VIII.C)

Materials to Be Supplied by the User

• water bath or heating block, preheated to 70°C• water bath or heating block, preheated to 42°C• water bath or heating block, preheated to 37°C• 80% ethanol, molecular biology grade• microcentrifuge• spectrophotometer• microcuvette• Cy®3 dCTP (Amersham Biosciences, Ltd. #PA53021)• Cy®5 dCTP (Amersham Biosciences, Ltd. #PA55021)

Materials Supplied in the Following Systems: 40051–40056

A. cDNA Labeling Protocol for Total RNA

1. For each Cy®-dye labeling reaction, assemble the following reagents in amicrocentrifuge tube. Keep the reagents on ice, and mix the total RNA andprimers as follows:

Total RNA or Total RNA Positive Control 5µgRandom Primers (3µg/µl) 1µlOligo(dT) Primer (2µg/µl) 1µl

Nuclease-Free Water to a total volume of 20µl

2. Incubate RNA/primer solution at 70°C for 10 minutes, then place on ice.



Never ...store the ChipShot™Reverse Transcriptase atroom temperature.

!

Protect...labeled dNTPs fromlight at all times to preventphotobleaching.

!

ChipShot™ Labeling System• MgCl2 (25mM)• Oligo(dT) Primer• dNTP Mix, Total RNA• dNTP Mix, mRNA• Random Primers• ChipShot™ Reverse

Transcriptase 5X ReactionBuffer

• ChipShot™ ReverseTranscriptase

• Nuclease-Free Water

• Total RNA Positive Control• RNase H• RNase Solution (RNase A)

ChipShot™ Labeling Clean-UpSystem• Binding Solution• Binding Particles• Spin-X® Columns• Elution Buffer• Collection Tubes• Sodium Acetate, 3M (pH 5.2)

For technical assistancewith cDNA labeling,please contact your localPromega Branch Office or distributor. Contactinformation available at:www.promega.com


Note: The Total RNAPositive Control is provided as a control forthe labeling and clean-up procedures.



Note: Cy®-dye stocks aretypically supplied as25nmol in 25µl. This is a1mM stock.

Note: Cy®-dye stocks aretypically supplied as25nmol in 25µl. This is a1mM stock.

The dNTP...mixes for mRNA and total RNA are NOT inter-changeable and are opti-mized for use with Cy®-labeled dCTP. Do not usedUTP.

!

The dNTP...mixes for mRNA and total RNA are NOT inter-changeable and are opti-mized for use with Cy®-labeled dCTP. Do not uselabeled dUTP.

!3. While the RNA/primer solution is incubating at 70°C, prepare labeling mix

as follows. Perform Cy®3 and Cy®5 reactions in separate tubes.

Component Cy®3 Cy®5ChipShot™ Reverse Transcriptase

5X Reaction Buffer 8µl 8µlMgCl2 (25mM) 4.8µl 4.8µldNTP mix, Total RNA 2µl 3µlCy®3 dCTP (1mM) 1µl –Cy®5 dCTP (1mM) – 1µlChipShot™ Reverse Transcriptase 3.2µl 3.2µlNuclease-Free Water 1µl –final volume 20µl 20µl

4. Add the entire 20µl labeling mix to each tube of RNA/primer solution, vortex,spin briefly and incubate at room temperature (22–25°C) for 10 minutes pro-tected from light.

5. Incubate at 42°C for 2 hours protected from light.

6. Add 1.0µl RNase H and 0.35µl RNase Solution to each cDNA-synthesis reac-tion. Mix gently and incubate at 37°C for 15 minutes.

B. cDNA Labeling Protocol for mRNA

1. For each Cy®-dye labeling reaction, assemble the following reagents in amicrocentrifuge tube. Keep the reagents on ice, and mix the mRNA andprimers as follows:

mRNA 1.5µgRandom Primers (3µg/µl) 1µl

Nuclease-Free Water to a total volume of 20µl

2. Incubate RNA/primer solution at 70°C for 10 minutes, then place on ice.

3. While the RNA/primer solution is incubating at 70°C, prepare labeling mixas follows. Perform Cy®3 and Cy®5 reactions in separate tubes.

Component Cy®3 Cy®5ChipShot™ Reverse Transcriptase

5X Reaction Buffer 8µl 8µlMgCl2 (25mM) 4.8µl 4.8µldNTP mix, mRNA 2µl 3µlCy®3 dCTP (1mM) 1µl –Cy®5 dCTP (1mM) – 1µlChipShot™ Reverse Transcriptase 3.2µl 3.2µlNuclease-Free Water 1µl –final volume 20µl 20µl

4. Add the entire 20µl labeling mix to each RNA/primer solution, vortex, spinbriefly and incubate at room temperature (22–25°C) for 10 minutes pro-tected from light.

5 Incubate at 42°C for 2 hours protected from light.

6. Add 1.0µl RNase H and 0.35µl RNase Solution to each cDNA-synthesis reac-tion. Mix gently and incubate at 37°C for 15 minutes.

C. Purifying Cy®-Labeled cDNA

1. To 40µl of the synthesized, labeled cDNA, add the following components in the order listed:Binding Particles 18µlSodium Acetate, 3M (pH 5.2) 4µlBinding Solution 310µl

2. Vortex gently for 5–10 seconds to mix.

3. Incubate at room temperature for 1 minute.

4. Repeat Steps 2 and 3.

5. Apply entire contents of tube (solution and Binding Particles) to a Spin-X®

column and cap the tube.

6. Spin at 10,000 × g for 1 minute.

7. Discard the column flowthrough.

8. Wash column with 500µl of 80% ethanol, cap the tube, and centrifuge at10,000 × g for 60 seconds.

9. Discard the column flowthrough.

10. Repeat Step 8 and 9 twice for a total of 3 washes.

11. Centrifuge column at 10,000 × g for 1 minute to remove traces of ethanol.

12. Place column in a clean Collection Tube (provided).

13. To elute labeled cDNA, add 60µl of Elution Buffer and let the column standat room temperature for 1 minute. Centrifuge at 10,000 x g for 1 minute, dis-card column. The eluted cDNA can be stored in a light-proof container at4°C for several weeks.

14. Quantitate absorbance at 260, 550 and 650nm, and calculate frequency ofincorporation.

Absorbance readings should be taken using undiluted cDNA directly in a micro-cuvette. Diluting the cDNA prior to reading the absorbance may give inaccuratereadings due to low concentration. The cDNA used for spectrophotometry shouldbe recovered for use in the hybridization reaction. Clean the cuvette thoroughlybetween samples to prevent cross-contamination.

Frequency of incorporation (FOI) is defined as the number of Cy®-labelednucleotides incorporated per 1,000 nucleotides of cDNA. Best results areobtained with cDNAs having an FOI as recommended in Table 2. The FOI can becalculated as follows:

FOI = pmol of dye incorporated × 324.5ng of cDNA

Amount of labeled cDNA (ng) = A260 × 37 × total volume (µl)

For Cy®3: pmol of dye incorporated = A550 x total volume (µl)0.15

For Cy®5: pmol of dye incorporated = A650 × total volume (µl)0.25



Resuspend...the Binding Particlesthoroughly before pipetting.

!

For technical assistancewith labeled cDNA purifi-cation, please contactyour local PromegaBranch Office or distributor. Contactinformation available at:www.promega.com


Protect ...the labeled sample fromlight as much as possible.

!

Keep tube...capped during all washsteps.

!

Note: These equations were generated using the following constants: Average Molar Mass ofdNTP = 324.5; One A260 unit of single-stranded DNA = 37µg/ml; Extinction Coefficient of Cy®3 =150,000M-1cm-1 at 550nm; Extinction Coefficient of Cy®5 = 250,000M-1cm-1 at 650nm.

To further assess the quality of the labeling reaction, and to improve repro-ducibility and validity of data generated, “yield ratio” should also be calculated(see Table 2).Yield ratio is calculated as: FOI x 100

ng DNA

and is expressed arbitrarily as a unitless percentage. The ratio of FOI-to-yieldfrom a labeling reaction better assesses quality than FOI alone because it high-lights divergence in yield and incorporation rate. For example, a reaction thathad a defective RNase H step might show an increased yield and decreasedFOI due to the presence of unlabeled RNA. The values for yield and FOI mightbe within the recommended values but on the high and low ends, respectively.However, the yield ratio for this example would be unacceptably low. Estimatingthe yield ratio guards against generating compromised data.



Best results are obtained with cDNAs that fall into the following ranges:

Table 2. Expected cDNA Yields and FOI.

5µg total RNA ng yield pmol FOI FOI:Yield RatioCy®3 1,200–2,400 100–170 20–35 1.25%–2.7%Cy®5 900–2,400 45–120 12–25 0.60%–1.5%

1.5µg mRNA ng yield pmol FOI FOI:Yield RatioCy®3 350–650 40–75 25–45 5.5%–11%Cy®5 325–650 20–50 15–35 4%–6%

VI. Hybridization Protocol

Materials to be Supplied by the User

• slide coverslips (Corning Cat.# 2870 and 2940)• speed vacuum concentrator• water bath or heat block, preheated to 42°C• water bath or heat block, preheated to 95°C• microcentrifuge• Corning® Hybridization Chamber (Cat.# 2551)• staining jar (e.g., TPX VWR International Cat.# 25460-907)• light-proof container for slide storage• deionized water (18 MegaOhm MilliQ® preferred)

Materials Supplied in the Following Systems: 40051–40056

• Pronto!™ Universal Pre-Soak Solution• Pronto!™ Universal Pre-Hybridization Solution• Pronto!™ Universal Hybridization Solution• Universal Wash Reagent A• Universal Wash Reagent B• Pre-Soak Tablets

A. Preparation of Wash Solutions

The volumes of Universal Wash Reagents A and B provided in the Pronto!™Universal Validation and Hybridization Kits are sufficient for processing 10 and25 arrays. We recommend preparing wash solutions at one time as described inorder to control variation in the preparation. The following volumes for wash solu-

For technical assistancewith hybridization, pleasecontact CorningIncorporated,Life Sciences. Contactinformation available at:www.corning.com/lifesciences


tion preparation are for 10 microarrays and should be adjusted by multiplying by2.5 for the Hybridization Kit (25 arrays). Carefully follow the order of addition.

Wash Solution 1deionized water (18 MegaOhm Milli-Q® preferred) 447.5mlUniversal Wash Reagent A 50mlUniversal Wash Reagent B 2.5ml

Wash Solution 2deionized water (18 MegaOhm Milli-Q® preferred) 1,425mlUniversal Wash Reagent A 75ml

Wash Solution 3Wash Solution 2 300ml deionized water (18 MegaOhm Milli-Q® preferred) 1,200ml

B. Pre-Soak and Pre-Hybridization

Note: We recommend processing 5 slides in a TPX staining jar (VWR Int. Cat.#25460-907), which requires 100ml of buffer for each step in the following protocol.If you use an alternative wash vessel, please adjust your volumes accordingly.

1. Heat required volumes of both Pronto!™ Universal Pre-Soak Solution andPronto!™ Universal Pre-Hybridization Solution to 42°C for at least 30 minutes.

2. Add 1 Sodium Borohydride Pre-Soak Tablet to 100ml of 42°C Universal Pre-Soak Solution. Allow the tablet to dissolve completely (about 5 minutes).

3. Immerse arrays in solution from Step 2 and incubate at 42°C for 20 minutes.

4. Transfer arrays to Wash Solution 2 and incubate at ambient temperature for30 seconds.

5. Transfer to a fresh container of Wash Solution 2 for 30 seconds.

6. Transfer arrays to 42°C Universal Pre-Hybridization Solution (fromStep 1) and incubate for 15 minutes.

7. Transfer arrays to a fresh container of Wash Solution 2 and incubate atambient temperature for 1 minute.

8. Transfer arrays to Wash Solution 3 and incubate at ambient temperature for30 seconds.

9. Transfer arrays to a fresh container of Wash Solution 3 and incubate atambient temperature for 30 seconds.

10. Dip arrays in nuclease-free water at ambient temperature (22–25°C), anddry by blowing high-purity nitrogen gas over the array or by centrifugation at1,600 × g for 2 minutes.

Note: 50ml conical tubes may be used for centrifugation; place 1 array inthe conical tube, and then centrifuge. Use a balance of tubes when cen-trifuging, and drop the arrays into the conical tube using forceps on the bar-coded side. If a rack is used, ensure that the rack is secured on the spin-ning, platform and use a proper balance.

11. Store slides in a dust-free environment (or in slide holder) until ready for use.

Note: Slides should be stable for several weeks if stored in a dust-free, non-oxidizing environment.



20 minutes isrequired for pre-

soak, but extending thetime beyond 20 minutes isNOT advisable and mayreduce signal.

!

DO NOT...add Sodium Boro-hydride Pre-Soak Tabletmore than 15 minutesbefore use.

Note: Arrays should befree of all residual solutionbefore hybridization. Besure to perform all washesas indicated.

Note: If using nitrogen gas,blow-dry the back of thearray first and then thefront. Blow dry as quicklyas possible. If centrifuga-tion is used, transport thewash containers with thearrays to the centrifugeduring the final wash step.

DO NOTlet arrays dry out

between wash steps,because this will causehigh backgrounds.



C. Hybridization

1. Wash the required number of pieces of cover glass (at least 1 piece of coverglass per array should be processed) with nuclease-free water, followed byethanol. Dry cover glass by blowing high-purity compressed nitrogen gas orallow to air-dry in a dust-free environment.

2. Dry the appropriate amount of each dye-labeled cDNA using a speed-vacuum concentrator, then dissolve the cDNA in the required volume ofPronto!™ Universal Hybridization Solution.

Calculating the Volume of Hybridization Solution to Use

The volume of Pronto!™ Universal Hybridization Solution needed depends on thesize of the printed area and cover glass. For regular cover glass, such as the recommended Corning® 2870 and 2940 cover glass product lines, use 2.5–3.5µlof hybridization solution per cm2 of surface area. This range of volume will accom-modate differences in humidity conditions and hybridization times. The fluores-cence strength required to achieve high levels of sensitivity and broad dynamicrange depends on the template used to synthesize the Cy®-cDNA.

Calculating the Amount of cDNA to Use

Total RNA. For Cy®-labeled cDNA made from total RNA, dry down 1.0pmol oflabeled cDNA per microliter of hybridization solution that will be used per dye.

mRNA. For Cy®-labeled cDNA made from mRNA, dry down 0.25pmol of incorpo-rated nucleotides per microliter of hybridization solution that will be used per dye.

3. Incubate the labeled cDNA solution at 95°C for 5 minutes, protecting sam-ples from light.

4. Centrifuge the cDNA at 13,500 × g for two minutes to collect condensation.Do not place the solution on ice because this will cause precipitation ofsome of the components.

5. Place array in Corning® Hybridization Chamber (Cat.# 2551). Pipet thelabeled cDNA gently up and down and then transfer onto the surface of theprinted side of the slide. Carefully place the cover glass on the array. Avoidtrapping air bubbles between the array and the cover glass. Small air bub-bles that do form usually dissipate during hybridization. Assemble the cham-ber as described in its package insert.

6. Incubate the chamber-array assembly at 42°C for 14–20 hours using awater bath or a hybridization oven.

Table 3. Examples of Recommended Hybridization Solution Volumes and pmolcDNA Based on Varying Coverslip Sizes.

Amount of Amount ofVolume of Labeled cDNA Labeled cDNA

Coverslip Surface Area Hybridization from Total RNA from mRNASize cm2 Solution (per slide) (per slide)

22 × 22mm 4.84 12–17µl 12–17pmol 3–4pmol24 × 40mm 9.60 24–33µl 24–33pmol 6–8pmol24 × 60mm 14.4 36–50µl 36–50pmol 9–12pmol*If doing a two-color hybridization, combine the recommended amount of both dye-labeled cDNAs. For example, for a 22 × 22mm coverslip with a two-color hybridization using total RNA-derived cDNA, combine12–17pmol of Cy®3-labeled and 12–17pmol of Cy®5-labeled cDNAs.

Note: Hybridizations longerthan 20 hours are not rec-ommended.



D. Post-Hybridization Washes

Note: We recommend processing 5 slides in a TPX staining jar (VWR Int., Cat.#25460-907), which requires 100ml of buffer for each step in the following proto-col. If you choose to use an alternative wash vessel, please adjust your volumesaccordingly.

Do not allow the arrays to dry out between washes, as this irreversibly increasesbackground levels. Multiple containers are needed to perform the washes in themost efficient manner. Have all containers and the volumes of washing solutionsready before starting the procedure.

1. Heat required volume of Wash Solution 1 to 42°C for at least 30 minutes(note that Steps 3 and 4 both require prewarmed solutions).

2. Disassemble the hybridization chambers with the printed array side facing up.

3. Immerse arrays in Wash Solution 1 at 42°C for 1–2 minutes until the coverglass falls from the slide.

4. Transfer arrays to a fresh container of Wash Solution 1 at 42°C and incubatefor 5 minutes.

5. Transfer arrays to Wash Solution 2 at ambient temperature (22–25°C) andincubate for 10 minutes.

6. Transfer arrays to Wash Solution 3 at ambient temperature and incubate for2 minutes.

7. Repeat Step 6 twice.

8. Dry arrays by blowing clean compressed nitrogen gas or by centrifugation at1,600 × g for 2 minutes.

Note: 50ml conical tubes may be used for centrifugation; place 1 array inthe conical tube, and then centrifuge. Use a balance of tubes when cen-trifuging, and drop the arrays into the conical tube using forceps on the bar-coded side. If a rack is used, ensure that the rack is secured on the spinningplatform, and use a proper balance.

9. Store arrays in light-proof container until ready to scan.

DO NOT...let arrays dry outbetween wash steps. Thisirreversibly increasesbackground, and data willbe lost.

Note: Always wash arrayshybridized with differentprobes separately and withfreshly prepared washsolutions.

Note: If using nitrogen gas,blow-dry the back of thearray first and then thefront. Blow dry as quicklyas possible. If centrifuga-tion is used, transport thewash containers with thearrays to the centrifugeduring the final wash step.



VII. Troubleshooting

A. Troubleshooting (Printing)

Symptoms Possible Causes CommentsIrregular spot mor- Bent or broken Check physical integrity of thephology and position printing pins pins regularly using a microscope.

Replace bent or broken pins.Replace pins that have exceededtheir useful life. Follow manu-facturer’s recommendations.

Buildup of salts and Clean printing pins thoroughlyother materials on pins after each run. Observe manu-

facturer’s recommendations.Optimize pin washing and dryingsteps of printing program forapplication at hand. Nonaqueousprinting solutions may require amore rigorous pin washing thanaqueous solutions.

Spotting Solution volume Identify and replace “thirsty” pins.difference among wells of Well volume that decreases morea source plate due to quickly in a few wells may indicate“thirsty” pins that the printing pin visiting those

wells is taking up more of thesolution than other pins, hence theterm “thirsty” pin.

Spotting Solution volume Optimize humidity for printing.difference among wells of Reduce number of freeze-thaw a source plate due to cycles.evaporation Keep print run as short as possible.

Pronto!™ Spotting Solution is de-signed to reduce the amount of evaporation and therefore reducethe influence of evaporation on source plate well volumes.

Adding detergents or Use Pronto!™ Spotting Solutioncontaminants such as as provided. Do not add detergentspolysaccharides to or other chemicals to the SpottingPronto!™ Spotting Solution. Pronto!™ SpottingSolution Solution is quality tested and ready

to use as a 1X solution. Detergentsand contaminants reduce DNAbinding to the UltraGAPS™surface.

Running spots due to Remove potential sources of sur-particulates on surface of face particulates after opening slide or pins package. Do not use powdered

gloves while handling slides. Donot place arrayer near ventilationducts. Clean dusty printing areas.

For technical assistancewith printing, please con-tact Corning Incorporated,Life Sciences.Contact informationavailable at:www.corning.com/lifesciences


Additional TroubleshootingInformation is available at:www.prontosystems.com/na/en/products/Protocols_Troubleshooting



Troubleshooting (Printing), continued

Symptoms Possible Causes CommentsDoughnut-shaped Contact-point deposi- Occurrence and negative effects ofspots tion (CPD) due to slide CPD can be minimized by main-

surface damage taining printing chamber humidityabove 45%, reducing the strike force of the printing pins and re-ducing particles in the printingenvironment and during slidehandling. CPD is a phenomenoninvolving the anchoring of the liquidcontained in a drying dropletonto surface irregularities. Re-ducing pin-strike force minimizespotential physical damage to theaminosilane-coated substrate andthe formation of deep anchoring sites for the spotted liquid.

Spots with “comets” or Loosely bound DNA Reduce DNA concentration in“tadpoles” reattaches to adjacent printing solution. Make sure that

reactive areas on the DNA concentration is optimized.slide surface Increase energy input during

immobilization of the spotted DNA(UV crosslinking or baking).Dry arrays by incubating at 65°Cfor 5–10 minutes immediatelybefore immersing in Pre-Hybridization Buffer. Upon contactwith hybridization solutions,loosely bound DNA lifts and re-attaches to adjacent reactive areas.Increasing energy reduces the reactivity of the unused surface.

Printed areas not dry Dry array completely before be-before hybridization ginning hybridization process.

Undried DNA can “drift” from theprinted spot resulting in thin tailsand uninterpretable data.

DNA content carry- Incomplete washing Optimize pin wash cycles duringover (cross-talk) during of printer pins between printing to make sure no carryoverprinting (positive signal samples takes place.in negative controlspots)



B. Troubleshooting (RNA Isolation)

Symptoms Possible Causes CommentsLow A260/A280 ratios Protein contamination Several methods may be used for

in RNA preparation removing contaminating proteinfrom RNA solutions. The mostexpedient method is to perform aphenol:chloroform extraction. Thisorganic extraction should yield higher A260/A280 ratios.However, loss of RNA (up to 40%)should be expected.

Guanidine isothiocyanate Precipitate the RNA by adding contamination in RNA NaOAc to a final concentration ofpreparation 0.1M. Add 2.5 volumes of ethanol.

Incubate for 30 minutes at –20°C.Collect the RNA by centrifugation at 10,000 × g for 15 minutes at 4°C. Resuspend the RNA in nuclease-free water.

Low A260 Tissue or homogenized Tissues or cell lysates that have(low yield) lysate stored at –20 been stored frozen may have

or –70°C less total RNA. For optimal performance, purify the RNA as soon as the lysate is prepared.

Tissues contain low Tissues and cells vary in totalamounts of total RNA amount of RNA that can be

purified based on wet weight. Iftotal RNA yields are low, increase the amount of starting materialthat is processed.

Sample integrity poor Samples that were not homo-genized or frozen immediatelyupon isolation may have de-creased amounts of RNA andreduced RNA integrity. Freezetissues immediately in liquidnitrogen and store at –70°C if theycannot be immediately processed.Homogenized samples should bestored at –20 or –70°C.

For technical assistancewith RNA isolation, pleasecontact your localPromega Branch Office or distributor. Contactinformation available at:www.promega.com




Troubleshooting (RNA Isolation), continued

Symptoms Possible Causes CommentsLow A260 Exceeded binding If the lysate contains more RNA(low yield) capacity of the than the capacity of the Spin(continued) membrane in the Spin Basket, the excess RNA will be

Basket washed away during the washsteps. When maximum recovery isessential, divide the lysate and perform multiple purifications. Poolthe resulting RNA solutions, and determine the total yield. The maximum lysate capacity of theSpin Baskets is 175µl/column.

Homogenate not heated Heat homogenate at 70°C for 3at 70°C (Section IV.C, minutes for optimal recovery of Step 5; IV.D, Step 9, IV.E, total RNA. Failure to heat this mix-Step 6; IV.F, Step 5) ture will decrease yields.Steps not followed The SV Total RNA Isolation Systemcorrectly or wrong is a multiple-step procedure thatreagents used requires the correct reagents in

the correct order. This ensures thatthe RNA remains bound to themembrane during purification.Wizard® Plus SV DNA PurificationBuffers are not compatible with thissystem and should not be used.

Ethanol not added to Prepare the solutions as the SV DNase Stop instructed in Section IV.A beforeSolution or the SV beginning the procedure. Mark theRNA Wash Solution bottle to record ethanol addition.Lysate allowed to over- Work as quickly as possible.heat during homo- Lysates can be placed on icegenization during sample preparation.

Use ice-cold SV RNA Lysis Bufferto improve yield and stability ifoverheating is a problem. Verifythat the homogenizer head is properly covered with lysateduring homogenization.

Low concentration Small sample input Purified total RNA can be concen-trated by precipitating the RNA andresuspending in a smaller volume.Add NaOAc to a final concentra-tion of 0.1M. Add 2.5 volumes of95% ethanol. Incubate 30 minutesat –20°C. Collect RNA by centri-fuging at 10,000 × g for 15 minutesRemove supernatant. Wash pelletwith 70% ethanol. Centrifuge at10,000 × g for 10 minutes to repel-let RNA. Remove supernatant. Re-suspend in nuclease-free water.



Troubleshooting (RNA Isolation), continued

Symptoms Possible Causes CommentsClogged Spin Baskets Lysate too concentrated If the homogenized lysate is dif-

ficult to pipet easily, the cell lysateis too concentrated, and the SVRNA Dilution Buffer will noteffectively clear the lysate. Concen-tration of RNA will vary betweentissues. If the lysate is too viscous,dilute with SV RNA Lysis Bufferbefore adding SV RNA Dilution Buffer. Use no more than 175µl of lysate per prep.

Pellet disturbed after Carefully pipet the cleared lysateclearing the lysate after the centrifugation step. Avoid

disturbing the pellet of precipi-tated proteins and cellular debris.

Lysate too viscous to Initial lysate too viscous Dilute lysate with SV RNA pipet easily Lysis Buffer.

Lysate becomes too Further homogenize sample toviscous while sitting on shear genomic DNA. Over-homo-ice genization of lysates can result in

lower RNA yields; only homo-genize as much as necessary.

RNA degradation RNase introduced by Use DEPC-treated glassware andhandling solutions and disposable plastic-

ware when manipulating andstoring RNA. Wear gloves at alltimes. RNases introduced afterelution will degrade the RNA.

RNA degraded during Work quickly during samplesample preparation preparation. (See comments under

Low A260: Sample integrity poor.)

DO NOT...treat Tris buffers

with DEPC. DEPC reactsrapidly with amines andcannot be used to treatTris buffers.

C. Troubleshooting (cDNA Labeling and Clean-Up)

Symptoms Possible causes CommentsLow yield of cDNA RNA degradation/ Always use nuclease-free,

RNase introduced commercially autoclaved reactionduring handling tubes, sterile aerosol-barrier tips,

and gloves.Ensure that reagents, tips andtubes are kept RNase-free by using sterile technique.RNA storage conditions are veryimportant. Store at –70°C. KeepRNA target in single-use aliquotsto minimize freeze-thaw cycles.Once thawed, keep RNA on ice.



For technical assistancewith cDNA labeling,please contact your localPromega Branch Office or distributor. Contactinformation available at:www.promega.com


C.Troubleshooting (cDNA Labeling and Clean-Up), continued

Symptom Possible Causes CommentsLow yield of cDNA RNA degradation/ Use RNasin® Ribonuclease (continued) RNase introduced Inhibitor (Promega Cat.# N2511,

during handling N2515) to inhibit degradation of (continued) target during cDNA synthesis

(40 units/40µl reaction).Use DEPC-treated glassware andsolutions and disposableplasticware when manipulatingand storing RNA. Wear gloves at alltimes. RNases introduced afterelution will degrade the RNA.Work quickly during samplepreparation. (See Section VII.BLow A260: Sample integrity poor.)

cDNA degradation DNase contamination from the RNA preparation method may bedigesting the cDNA. Carefullyfollow the SV Total RNA Isolationprotocol provided in this manualto completely remove theDNase I (Section IV).

Inhibitors present in Inhibitors such as SDS, EDTARNA preparation polysaccharides, heparin,

guanidine isothiocyanate or othersalts may carry over from some RNA preparations and interfere with cDNA labeling. Isolate RNAusing the SV Total RNA Isolation Protocol provided in Section IV.To detemine if the experimentalRNA preparation contains aninhibitor, set up a spiking experiment by adding controlRNA to experimental RNA, andassess inhibition of cDNAsynthesis.

Incorrect primer:RNA Use the recommended primerratio concentration and the recom-

mended amount of template RNA.Mix well before use.

Incorrect dNTP concen- The dNTP mixes for mRNA andtration total RNA are NOT interchangeable.

Confirm that the appropriate dNTPmix is used for the type of RNAtemplate in the reaction.

Insufficient reverse Use the recommended amount oftranscriptase activity ChipShot™ Reverse Transcriptase

in the labeling reaction.



C.Troubleshooting (cDNA Labeling and Clean-Up), continued

Symptoms Possible Causes CommentsLow yield of cDNA Reverse Transcriptase Be sure to combine the reverse(continued) inactivated before cDNA transcription reaction mix with

synthesis the target AFTER thermal de-naturation. Thermal denaturationwill inactivate the ChipShot™Reverse Transcriptase. Storeenzyme at –20°C.

Insufficient mixing of Vortex all reagents (exceptreagents enzymes) before use.Insufficient binding of Perform recommended mixingthe cDNA to the before washing the BindingBinding Particles Particles to allow maximum binding

of cDNA to Binding Particles.Poor elution of the Perform recommended ambientcDNA from the temperature incubation of the elu-Binding Particles tion buffer with the cDNA/Binding

Particles before the elution spin.Cy®-labeled dUTP used The dNTP mixes are optimized for

use with Cy®-labeled dCTP. UsingCy®-labeled dUTP will result inextremely poor yield and low FOI.

cDNA yield is Insufficient RNase Residual RNA present in cDNAacceptable, but activity sample can result in A260 absor-Frequency of bance reading that does not ac-Incorporation (FOI) curately represent the amount ofis low labeled cDNA present. Perform re-

commended treatment step beforeBinding Particle clean-up. Refer toTable 2, Section V.C for expectedyield ratios.

Poor Cy®-labeled Insufficient concentration of Cy®-nucleotide incorporation labeled dCTP. Use recommended

amount of Cy®-labeled nucleotide.Protect Cy®-dyes from heat andlight to ensure that Cy® dyes do not degrade, especially Cy®5 dye.

Inconsistent yield Inconsistent amounts of Resuspend Binding Particles justBinding Particles used in before making aliquots. Repeatpost-labeling clean-up mixing while making aliquots.

cDNA yield is acceptable Contaminating Cy®- Follow the cDNA purification but Frequency of labeled nucleotides protocol (Section V.C).Incorporation (FOI)is high

For technical assistancewith labeling and purifyingCy®-labeled cDNA, pleasecontact your localPromega Branch Office or distributor. Contactinformation available at:www.promega.com




For technical assistancewith hybridization, pleasecontact CorningIncorporated, Life Sciences. Contactinformation available at:www.corning.com/lifesciences


Additional TroubleshootingInformation is available at:www.prontosystems.com/na/en/products/Protocols_Troubleshooting

D. Troubleshooting (Hybridization)

Symptom Possible Causes CommentsSpots appear smeared DNA concentration too Print using lower DNA concentra-as comets high tion. Make note of UV energy

required for cDNA and longoligonucleotides.

UV lamp does not Measure and calibrate UV lampimmobilize DNA output. Bake slides 2–4 hourseffectively in an 80°C oven.Coverslip slid into place Drop coverslip squarely on theover labeled cDNA array. Do not slide coverslip acrosssolution the array. Practice coverslip place-

ment with water and plain slidebefore performing on an array.

Insufficient blocking Pre-hybridize for 15 minutes.Printing Problems See Section VIII.A for comments.

Low fluorescent Too little or too much Check the FOI of cDNAsignal incorporation of before using for hybridization.

fluorescent dye in If the appropriate dye incorpora-sample probe tion is obtained, less labeled cDNA

is used, and the highest signalsand lowest background areattained (Table 2, Section V.C).

Degradation of the Keep labeled dyes and labeled fluorescent dye in cDNA cDNA protected from light.

Fluorescent spots in Powder from gloves con- Use powder-free gloves duringthe background taminates slides during all portions of the microarray

printing or hybridization process.Dust settles on slide Work in a dust-free hood or

environment.Uneven or high Air bubbles trapped under Small bubbles dissipate during background coverslip during hybridi- hybridization; no action necessary.

zation prevent labeled Hold slide and coverslip so thatcDNA from contacting larger bubbles rise to the top ofthe arrayed nucleic acid the slide and escape from under

the coverslip. Practice coverslipplacement with water and a plainslide before performing on an array.

Incomplete washing Use clean washing vessels foreach run. Washing steps are critical to low backgrounds. Slidesshould not be allowed to dry untilfinal wash step.

Incomplete or improper Immediately blow-dry or spin-drydrying slides after last wash step.Excess amounts of Check FOI of labeled cDNA beforelabeled cDNA with poorly using it for hybridization. Refer toincorporated dye Table 2, Section V.C for labeling

assessment guidelines.Improper slide storage Store slides after printing in

Corning® slide holder placed insealed plastic bag with #3 desiccant.



D. Troubleshooting (Hybridization), continued

Symptoms Possible Causes CommentsBlack holes Very low expressers Depending upon the appearance

surrounded by back- of the background around the blackground fluorescence hole, refer to appropriate back-

ground solutions (see above).Black holes appear as dark spotswithin the background field. Thesespots have the expected size,shape, and placement of printedspots.

Unexpected hybridi- Arrays hybridized with Always wash arrays hybridized withzation patterns different cDNAs washed different cDNAs separately and with

in same bath and wash freshly prepared wash solutions.solutions

Intense uniform fluore- Hybridization solution Insure that there is properscence around outer dried out humidity during hybridization.edge of coverslip If using Corning® Hybridization

Chamber (Cat.# 2551), add 10µlof deionized water to the humidifi-cation wells in the chamber base. Ifusing an alternative chamber, en-sure proper humidification.

- Be sure to drop coverslip into place. Sliding the coverslip into place can leave some cDNAuncovered on the arrayed slide.Be sure to match cDNA volume with coverslip size so that it doesnot wick to the underside of the slide. Refer to Table 3.

Insufficient blocking Ensure pre-hybridization occursfor 15 minutes.

Slides hybridized too long For longer hybridizations, useCorning® Hybridization Chambers(Cat.# 2551).

Irregular spot mor- Printing problems See Section VII.A for comments.phology and positionDoughnut-shaped Printing problems See Section VII.A for comments.spotsSpots with “comets” Printing problems See Section VII.A for comments.or “tadpoles”DNA content carry- Printing problems See Section VII.A for comments.over (cross-talk)during printing (postivesignal in negativecontrol spots)



VIII. Appendix

A. Composition of Buffers and Solutions

PBS buffer, 10X (per liter)

11.5g Na2HPO42g KH2PO4

80g NaCl2g KCl

Dissolve in 1 liter of sterile, deionizedwater. The pH of 1X PBS will be 7.4.

SV RNA Lysis Buffer

4M guanidine isothiocyanate0.01M Tris (pH 7.5)0.97% β-mercaptoethanol

(when added)

1X trypsin-EDTA solution

0.05% trypsin (w/v)0.53mM EDTA

Dissolve in 1X PBS.

SV DNase Stop Solution (concentrated)

5M guanidine isothiocyanate

10mM Tris-HCl (pH 7.5)

After dilution with ethanol, the final concen-tration (approximate) is 2M guanidineisothiocyanate, 4mM Tris-HCl (pH 7.5) and57% ethanol.

SV RNA Wash Solution(concentrated)

162.8mM potassium acetate27.1mM Tris-HCl

(pH 7.5 at 25°C)

After dilution with ethanol, the final concen-tration (approximate) is 60mM potassiumacetate, 10mM Tris-HCl (pH 7.5 at 25°C)and 60% ethanol.

Yellow Core Buffer

0.0225M Tris (pH 7.5)1.125M NaCl

0.0025% yellow dye (w/v)

Elution Buffer

10mM Tris-HCl (pH 8.5)



C. References

1. Bowtell and Sambrook (2003) DNA Microarrays: A Molecular CloningManual Cold Spring Harbor Laboratory Press.

2. Chirgwin, J.M. et al. (1979) Isolation of biologically active ribonucleic acidfrom sources enriched in ribonuclease. Biochemistry 18, 5294–9.

3. Adams, J. et al. (2002) Fast and Efficient Production of RNA with the T7RiboMAX™ Express System. Promega Notes 80, 5–9.

4. Ishida, S. et al. (2001) Role for e2F in control of both DNA replicationand mitotic functions as revealed from DNA microarray analysis. Mol.Cell Biol. 21, 4684–99.

5. Maleck, K. et al. (2000) The transcriptome of Arabidopsis thaliana duringsystemic aquired resistance. Nat. Genet. 26, 403–10.

6. Morgan, R.W. et al. (2001) Induction of host gene expression followinginfection of chicken embryo fibroblasts with oncogenic Marek’s diseasevirus. J. Virol. 75, 533–9.

Table 4. Average Yields of Total RNA Isolated from Tissues and Cells.

Maximum Average Average YieldAmount to Yield per per milligram Average Average

Samples Process Prep (µg) Tissue (µg) A260/A230 A260/A280

Mouse TissuesLiver 30mg 131 4.4 2.4 1.9Kidney 20mg 44 2.2 2.1 1.9Spleen 15mg 79 5.3 2.3 1.9Muscle 30mg 22 0.73 1.8 2.1Brain 60mg 39 0.65 2.1 2.1Rat TissuesPancreas 30mg 100 3.5 2.2 1.9Heart 60mg 16 0.27 1.8 2.1Lung 60mg 36 0.60 2.0 2.1Cell Line (RAW264.7)

5 x 106 cells 51 N/A 2.0 2.1Cell Line (HeLa)

2 × 106 cells 35 N/A 1.5 2.1Cell Line (293T)

2 X106 cells 15 N/A 1.5 2.1

N/A: Not applicable.

The Table 4 values represent means of results achieved at Promega.Yields will depend on thetype of tissue, cultures and metabolic state of the sample. The means for the cell line andspleen sample are the average of two and three determinations, respectively. HeLa cell datareflects the average of 16 isolations. 293T cell data relfects the average of 38 isolations. Themeans for all other samples are the average of at least six determinations.

Means were calculated using both the spin and vacuum formats for all rat and mouse tissuesamples listed, except kidney, liver, spleen and cell line. The cell line used was RAW264.7, amouse macrophage line grown to confluence in Dulbecco's modified Eagle medium plus 10%fetal bovine serum and 1mM pyruvate.

B. Average Expected Yields for RNA Isolations



7. Travers, K.J. et al. (2000) Functional and genomic analyses reveal anessential coordination between the unfolded protein response and ERassociated degradation. Cell 101, 249–58.

8. Ferbeyre, G. et al. (2000) PML is induced by oncogenic ras and promotes premature senescence. Gen. Dev. 14, 2015–27.

D. Related Products, Promega Corporation

Product Size Cat.#PolyATtract® mRNA Isolation System II 3 isolations Z5200with Magnetic StandPolyATtract® System 1000 with Magnetic Stand(e) scalable Z5420RNAgents® Total RNA Isolation System scalable Z5110For Laboratory Use.

E. Related Products, Corning Incorporated, Life Sciences

Product Size Cat.#96 Well UV Microplate 25/pk 3635

50/cs96 Well Half Area UV Microplate 25/pk 3679

50/cs384 Well UV Microplate 5/pk 3675

25/cs96 Well V-bottom Polypropylene Microplate 25/pk 3357

100/cs384 Well Polypropylene Microplate 25/pk 3656

100/csUltraGAPS™ Slides, with bar code 5/pk 40015

25/csUltraGAPS™ Slides, without bar code 5/pk 40016

25/csUltraGAPS™ Slides, with bar code (bulk pack) 25/pk 40017

25/csUltraGAPS™ Slides, without bar code (bulk pack) 25/pk 40018

25/csCover Glass, Square, No. 1½ 1oz. 2870

10oz.Cover Glass, Rectangle, No 1½ 1oz. 2940

10oz.Hybridization Chamber 1/pk 2551

5/csHybridization Chamber O-rings 5/pk 40001

5/cs96 Well Robolid-Rigid Cover 25/pk 3090with Attached Silicone Sealing Mat 50/cs384 Well Robolid-Rigid Cover 25/pk 3089with Attached Silicone Sealing Mat 50/csUniversal Lid-Rigid Lid for 25/pk 309996 and 384 Well Microplates 50/csAluminum Sealing Tape for 384 Well Blocks 100/pk 6569and Microplates 100/cs



Promega Corporation2800 Woods Hollow RoadMadison, WI 53711-5399 USA

Domestic:Toll Free in the USATel: 800-356-9526

International:Tel: 608-274-4330

Fax: 608-277-2516


InternetFor a comprehensive list of world wide offices, please visit:

www.prontosystems.com

Corning IncorporatedLife Sciences45 Nagog ParkActon, MA 01720 USA

Domestic:Toll Free in the USATel: 800-492-1110

International:Tel: 978-635-2200


Contact Information

NOTES



(a)U.S. Pat. Nos. 6,027,945 and 6,368,800, Australian Pat. No. 732756 and Japanese Pat. No. 3253638 have been issued to PromegaCorporation for methods of isolating biological target materials using silica magnetic particles. Other patents are pending.

(b)U.S. Pat. No. 6,218,531 and Australian Pat. No. 745185 have been issued to Promega Corporation for a method for isolating RNA. Otherpatents are pending.

(c)Australian Pat. No. 730718 and Singapore Pat. No. 64532 have been issued to Promega Corporation for an improved filtration system andmethod. Other patents are pending.

(d)Certain applications of this product are covered by patents issued and applicable in certain countries. Because purchase of this product doesnot include a license to perform any patented application, users of this product may be required to obtain a patent license depending upon theparticular application and country in which the product is used.

(e)U.S. Pat. No. 5,693,784 has been issued to Promega Corporation for methods for creating agglomerates from colloidal particles.

© 2003 Promega Corporation and Corning Incorporated. All Rights Reserved.

PolyATtract, RNAgents and Wizard are trademarks of Promega Corporation and are registered with the U.S. Patent and Trademark Office.ChipShot is a trademark of Promega Corporation.Corning, Discovering Beyond Imagination, Flame of Discovery design and Spin-X are registered trademarks of Corning Incorporated, Corning,NY. Pronto! and UltraGAPS are trademarks of Corning Incorporated, Corning, NY.

Cy is a registered trademark of Amersham Biosciences, Ltd. Milli-Q is a registered trademark of Millipore Corporation. RiboGreen is a registeredtrademark of Molecular Probes. Tissuemizer is a trademark of Tekmar Company.

All prices and specifications are subject to change without prior notice.

Product claims are subject to change. Please contact Promega Technical Services or access the Promega online catalog for the most up-to-date information on Promega products.

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