Indirect All-in-One Kit from Sample Prep to Hybridization

Overview

The BioModule™ Microarray Units provide qualified reagents and validated protocols for fluorescent labeling of cDNA synthesized from purified total RNA, and subsequent hybridization of the labeled cDNA to DNA microarrays for gene expression profiling (De Risi et al., 1996; Eisen & Brown, 1999).
 
The Indirect cDNA Labeling Unit includes the SuperScript™ Plus Indirect cDNA Labeling System for generating labeled cDNA using aminoallyl- and aminohexyl-modified nucleotides and either Alexa Fluor® 555 succinimidyl ester or Alexa Fluor® 647 succinimidyl ester.
 
The Direct cDNA Labeling Unit includes the SuperScript™ Plus Direct cDNA Labeling System for generating labeled cDNA using nucleotide mixes that contain Alexa Fluor® 555-aha-dUTP or Alexa Fluor® 647-aha-dUTP.
 
Both units include TRIzol® Reagent and the PureLink™ Micro-to-Midi Total RNA Purification Kit for high-quality, rapid purification of total RNA, and SuperScript™ III Reverse Transcriptase (RT) for high-temperature synthesis of first-strand cDNA from 5–20 µg of total RNA. Both units also include optimized microarray hybridization buffers that are compatible for use with Corning® GAPS, GAPS II, and UltraGAPS™ slides.
 
Summary of Components

Each BioModule™ Microarray Unit includes:

• TRIzol® Reagent and the PureLink™ Micro-to-Midi Total RNA Purification System, for isolating total RNA from mammalian cells, fresh and frozen tissues, whole blood, and liquid samples.
• Deoxyribonuclease I (DNase I), Amplification Grade, for eliminating DNA during RNA purification procedures
• SuperScript™ III Reverse Transcriptase for high-temperature cDNA synthesis, ensuring high specificity and high yields of cDNA as well as more full-length cDNA
• 5X First-Strand Buffer and anchored oligo(dT)20 primer for cDNA synthesis
• RNaseOUT™ Recombinant Ribonuclease Inhibitor, to safeguard against the degradation of target RNA due to ribonuclease contamination.
• Alexa Fluor®-labeled nucleotides or amine-modified nucleotides and Alexa Fluor® dyes, for labeling cDNA
• Spin cartridges and buffers for purifying the labeled cDNA
• Optimized array hybridization and wash buffers. For more information about each reagent

Labeling and Hybridization Workflow Overview

To use the BioModule™ Microarray Unit, you will:

• Purify total RNA from lysate using TRIzol® Reagent and the PureLink™ Micro-to-Midi Total RNA Purification System, and treat with DNase I
• Determine RNA quality and yield
• Synthesize and label cDNA using indirect or direct labeling
• Purify the labeled cDNA
• Hybridize the labeled cDNA to microarrays
• Analyze the results

Types of Products

Number of Boxes

The number of boxes provided with each unit is listed below

Shipping and Storage

The shipping conditions for each component are listed below. Upon receipt, store the components as described.

TRIzol® Reagent

This unit includes 100 ml of TRIzol® Reagent, which is sufficient for ~100 isolations.

PureLink™ Micro-to-Midi Total RNA Purification

The components provided with the PureLink™ Micro-to-Midi Total RNA Purification System are described below. Sufficient reagents are provided to perform 50 purifications.

DNase I, Amplification Grade
 
Two boxes of DNase I, Amplification Grade, are provided with each unit. Components are listed below. Sufficient reagents are provided for 40 on-column DNase I treatments.
 
Item                                                                         Amount
DNase I, Amp Grade                                             2 × 100 µl
10X DNase I Reaction Buffer                               2 × 100 µl
25 mM EDTA (pH 8.0)                                           2 × 100 µl

10X DNase I Reaction Buffer
10X DNase I Reaction Buffer contains 200 mM Tris-HCl (pH 8.4), 20 mM MgCl2, and 500 mM KCl.
 
Microarray Hybridization Solutions

The BioModule™ Microarray Hybridization Solutions are described below. Formulations are proprietary. Sufficient reagents are provided to perform 30 hybridizations. 

UltraPure™ Formamide

This unit includes 500 g of UltraPure™ Formamide in liquid form, which must be added to the Hybridization Solution before use.
 
SuperScript™ Plus Indirect cDNA Labeling System

The SuperScript™ Plus Indirect cDNA Labeling System is included with the BioModule™ Microarray Unit with Indirect cDNA labeling (Catalog no. WFGE03). Sufficient reagents are provided to perform 30 labeling reactions.

Introduction

This section provides guidelines and protocols for total RNA purification from animal cells or tissues using TRIzol® Reagent, the PureLink™ Micro-to-Midi Total RNA Purification System, and DNase I, Amplification Grade.
 
The protocols in this section use TRIzol® Reagent, the PureLink™ Micro-to-Midi Total RNA Purification System, and DNase I, Amplification Grade. Have all components from these boxes ready before proceeding.
 
Guidelines for Handling RNA

Follow the guidelines below to prevent RNase contamination and to maximize the RNA yield: 

• Use disposable, individually wrapped, sterile plastic ware.
• Use only sterile, disposable RNAse-free pipette tips and microcentrifuge tubes.  
• Wear disposable gloves while handling reagents and RNA samples to prevent RNase contamination from the surface of the skin. Change gloves frequently, particularly as the purification protocol progresses from crude extracts to more purified material.
• Always use proper microbiological aseptic techniques when working with RNA.
• Use RNase AWAY® Reagent to remove RNase contamination from work surfaces and non-disposable items such as centrifuges and pipettes used during purification.  

Guidelines for Sample Collection

Use the following guidelines for collecting your samples to minimize RNA degradation prior to RNA purification and to maximize the RNA yield:
 

• Always wear disposable gloves while handling samples and reagents to prevent RNase contamination.
• Work quickly during sample harvesting, use RNase-free dissection tools and containers (scalpels, dishes, tubes etc.) and work on RNAse-free work surfaces (use RNase AWAY® Reagent).
• To purify total RNA from fresh samples, keep fresh cell and tissue samples on ice immediately after harvesting; quickly proceed to adding RNA Lysis solution, sample lysis and homogenization.
• To purify total RNA from frozen samples, freeze samples immediately after harvesting in liquid nitrogen or on dry ice. Keep frozen samples at -80° or in liquid nitrogen until proceeding to sample lysis and homogenization.  
• Whole blood: Process freshly drawn blood immediately, and keep at room temperature until processing.

 
TRIzol® Reagent contains phenol and guanidine isothiocyanate. Toxic in contact with skin and if swallowed. Causes burns. Always wear a laboratory coat, disposable gloves, and eye protection when handling solutions containing this chemical. After contact with skin, wash immediately with plenty of detergent and water. Use in a chemical fume hood. Avoid breathing vapor. Do not add bleach or acidic solutions directly to solutions containing guanidine isothiocyanate or sample preparation waste. Guanidine isothiocyanate forms reactive compounds and toxic gases when mixed with bleach or acids.

Solutions containing ethanol are considered flammable. Use appropriate precautions when using these solutions. 

Use polypropylene tubes when working with TRIzol® Reagent. Do not use polystyrene tubes. 
 
Frozen tissue must remain frozen at –80°C prior to lysis. Cool tubes in dry ice before placing frozen tissue in them. Thawing of frozen tissue prior to lysis may result in RNA degradation and loss of RNA yield.
 
Additional Materials Needed:

You will need the following additional items not provided with this unit:

• DEPC-treated water
• Chloroform (molecular biology grade)
• 70% ethanol (in RNase-free water)
• 15-ml  and 1.5-ml RNase-free polypropylene microcentrifuge tubes  (Polypropylene is required for use with TRIzol® Reagent; do not use polystyrene tubes. Round-bottom 15-ml tubes are required for homogenization of tissues using a power homogenizer.)
• Microcentrifuge capable of centrifuging 12,000 × g
• Vortex mixer
• For tissue samples: Power homogenizer with a rotating tip that fits a 15-ml tube (e.g., Ultra Turrax® or Polytron® Homogenizer )
• 1.5 ml RNase-free tubes 
• RNase-free pipette tips

Preparing Wash Buffer II with Ethanol

Before using Wash Buffer II from the PureLink™ Micro-to-Midi Total RNA Purification System for the first time, add 60 ml of 96–100% ethanol directly to the bottle. Check the box on the Wash Buffer II label to indicate that ethanol was added.

Lysis and Homogenization of Cells

Follow the steps below to prepare lysates from up to 1 × 10⁶ cells:
 

1. Adherent cells: Lift cells from the plate by adding TE and resuspend in culture media. Count the cells, and transfer ≤1 × 10⁶ cells to a 15-ml polypropylene centrifuge tube.  Suspension cells: Transfer ≤1 × 10⁶ cells to a 15-ml polypropylene centrifuge tube.


2. Centrifuge at 2,000 × g for 5 minutes at room temperature to pellet the cells.


3. Carefully pipet the supernatant from the tube, leaving no more than 30 µl of supernatant. Take care not to disturb the cell pellet.


4. Immediately add 1 ml of TRIzol® to the tube. Vortex until the cell pellet is completely lysed and no visible particulate matter remains. If necessary, pipet the pellet up and down to disperse.


5. Transfer the lysate to a new, RNase-free 1.5-ml polypropylene microcentrifuge tube.

Proceed to Total RNA Purification Procedure (below).
 
Lysis and Homogenization of Tissues

Follow the steps below to prepare lysates from animal tissues. Use a power homogenizer with a rotating tip that fits a 15-ml round-bottom tube.
 
Note:    The volume of tissue should not exceed 10% of the volume of TRIzol® Reagent used for homogenization. Keep frozen tissues on dry ice until just prior to homogenization.
 

1. Add 1 ml of TRIzol® Reagent for every 50–100 mg of tissue to a 15-ml round-bottom polypropylene microcentrifuge tube. Immediately add fresh or frozen tissue to the tube and quickly homogenize the sample using a power homogenizer at a medium setting. Homogenize using short bursts for ~2 minutes. Avoid foaming by keeping the rotating tip submerged in solution while holding the tip against the tube wall. Keep homogenate on ice.


2. Transfer 1-ml aliquots of homogenate to separate 1.5-ml polypropylene microcentrifuge tubes on ice.


3. Centrifuge tubes at 2,000 × g for 5 minutes at 5°C to pellet cellular debris.


4. Carefully transfer the supernatant (~800 µl) containing the RNA from each tube to a new, RNase-free 1.5-ml polypropylene tube.


5. Add enough TRIzol® (~200 µl) to the tube to bring the total volume to 1 ml. Proceed to Total RNA Purification Procedure (below).

 
 
Total RNA Purification Procedure

Follow the steps below to purify your total RNA sample:

1. Add 0.2 ml of chloroform to each 1.5-ml tube from Step 5 of the lysis and homogenization protocols above. Mix well by vortexing.


2. Incubate at room temperature for 2 minutes to allow for phase separation.


3. Centrifuge the tube at 12,000 × g for 15 minutes at 5°C. After centrifugation, a clear aqueous upper phase and pink organic lower phase should be visible. Some white flocculent material may also be visible in the interface between the phases.


4. Using a pipette, very carefully remove the upper phase (up to 0.5 ml) without disturbing the lower phase or interface. Take care not to remove any flocculent material with the upper phase. (It is better to remove less of the upper phase if there is a danger of disturbing the lower phase or interface.) Transfer the upper phase to a new RNase-free 1.5-ml tube.


5. To the volume of upper-phase solution in the new tube, add an equal volume of 70% ethanol by adding ½ volumes sequentially, and mixing after each addition. Mix carefully to avoid precipitation of RNA due to local concentrations of ethanol.


6. Transfer up to 700 µl of the sample to a PureLink™ RNA Spin Cartridge pre-inserted in a collection tube. Centrifuge at 12,000 × g for 15 seconds at room temperature. Discard the flow-through, and re-insert the cartridge in the tube.


7. Repeat Step 6 until the entire sample has been processed.


8. Add 350 µl of Wash Buffer I to the spin cartridge. Centrifuge at 12,000 × g for 15 seconds at room temperature. Discard the flow-through, and re-insert the cartridge in the tube.


9. In a separate RNase-free tube, prepare a DNase I solution by adding:






10. Add the entire volume of DNase I solution directly onto the spin cartridge. Incubate at room temperature for 15 minutes.


11. Add 350 µl of Wash Buffer I to the spin cartridge. Centrifuge at 12,000 × g for 15 seconds at room temperature. Discard the collection tube.


12. Place the spin cartridge into a clean RNA Wash Tube, provided in the kit.


13. Add 500 µl Wash Buffer II with ethanol to the spin cartridge. Centrifuge at 12,000 × g for 15 seconds at room temperature. Discard the flow-through, and re-insert the cartridge in the tube.


14. Repeat Step 13 once.


15. Centrifuge the spin cartridge at 12,000 × g for 1 minute to dry the membrane with attached RNA. Discard the collection tube, and insert the cartridge into an RNA Recovery Tube supplied with the kit.


16. To elute the RNA, add 30 µl of RNase-free water to the center of the spin cartridge membrane, and incubate at room temperature for 1 minute.


17. Centrifuge the spin cartridge for 2 minutes at ≥12,000 × g  to collect the eluate.


18. Remove the recovery tube and add 10 µl of DEPC-treated water to the eluate. Add this solution back onto the spin cartridge membrane. Re-insert the cartridge in the recovery tube, and incubate at room temperature for 1 minute.


19. Centrifuge the spin cartridge for 2 minutes at ≥12,000 × g to collect the eluate containing the total RNA.
    Determine the quantity and quality of the RNA or store the purified total RNA at –80°C

1. Dilute an aliquot of the total RNA sample in 10 mM Tris-HCl, pH 7.5. Mix well. Transfer to a cuvette (1-cm path length). Note: The RNA must be in a neutral pH buffer to accurately measure the UV absorbance.


2. Determine the A260 of the solution using a spectrophotometer blanked against 10 mM Tris-HCl, pH 7.5. Calculate the amount of total RNA using the following formula:

• Vortex mixer
• Microcentrifuge
• Water baths, heating blocks, or incubator
• Aerosol resistant RNase-free pipette tips
• 1.5-ml RNase-free microcentrifuge tubes
• 1 N NaOH
• 1 N HCl
• 100% Isopropanol
• 100% Ethanol
• 75% Ethanol

1. Mix and briefly centrifuge each component before use.


2. Prepare each reaction as follows in a 1.5-ml RNase-free tube:
TOP



Component	Volume
5–20 µg purified total RNA	X µl
Anchored oligo(dT)20 primer (2.5 µg/µl)	2 µl
DEPC-treated water	to 18 µl


 

3. Incubate tubes at 70°C for 5 minutes, and then place on ice for at least 1 minute.


4. Add the following to each tube on ice:
   
   

   Component	Volume
   5X First-Strand buffer	6 µl
   0.1 M DTT	1.5 µl
   dNTP mix (including amino-modified nucleotides)	1.5 µl
   RNaseOUT™ (40 U/µl)	1 µl
   SuperScript™ III RT (400 U/µl)	2 µl
   Final volume	30 µl



5. Mix gently and collect the contents of each tube by brief centrifugation. Incubate tube at 46°C for 2­–3 hours. Note: A 3-hour incubation results in 20–30% higher cDNA yield than a 2-hour incubation.

1. Add 15 µl of 1 N NaOH to each reaction tube from Step 5, above. Mix thoroughly.


2. Incubate tube at 70°C for 10 minutes.


3. Add 15 µl of 1 N HCl to neutralize the pH and mix gently.

1. Add 700 µl of Binding Buffer (prepared with isopropanol as described on page 14) to the reaction tube containing the first-strand cDNA from Hydrolysis and Neutralization, Step 3, previous page. Vortex briefly to mix.


2. Each Low-Elution Volume Spin Cartridge is preinserted into a collection tube. For multiple reactions, clearly label each collection tube, and then load the cDNA/Binding Buffer solution directly onto the Spin Cartridge.


3. Centrifuge at 3,300 × g in a microcentrifuge for 1 minute. Remove the collection tube and discard the flow-through.


4. Place the Spin Cartridge in the same collection tube and add 600 µl of Wash Buffer (prepared with ethanol) as described on page 14) to the column.


5. Centrifuge at maximum speed for 30 seconds. Remove the collection tube and discard the flow-through.


6. Place the Spin Cartridge in the same collection tube and centrifuge at maximum speed for 30 seconds to remove any residual Wash Buffer. Remove the collection tube and discard.


7. Place the Spin Cartridge onto a new amber collection tube (supplied in the kit).


8. Add 20 µl of DEPC-treated water to the center of the Spin Cartridge and incubate at room temperature for 1 minute.


9. Centrifuge at maximum speed for 1 minute to collect the purified first-strand cDNA in the amber tube. The eluate contains your purified cDNA.   Proceed directly to Coupling the Fluorescent Dye below.

1. Dry the purified first-strand cDNA from Step 9, above, in a speed vac at medium heat until the volume is reduced to ~3 µl. Be careful not to overdry the sample.


2. Add 5 µl of 2X Coupling Buffer to the tube 


3. Add 2 µl of DMSO directly to a vial of Alexa Fluor® Reactive Dye to resuspend the dye. Vortex thoroughly and then spin briefly to collect the contents.


4. Add the DMSO/dye solution to the tube from Step 2 and vortex to mix thoroughly.


5. Incubate the tube at room temperature in the dark for 1–2 hours. The reaction can be stored overnight if necessary.

1. Add 700 µl of Binding Buffer (prepared with isopropanol) as described on page 14) to the reaction tube containing the labeled cDNA from Coupling Procedure, Step 5. Vortex briefly to mix.


2. Each Low-Elution Volume Spin Cartridge is preinserted into a collection tube. For multiple reactions, clearly label each collection tube, and then load the cDNA/Binding Buffer solution directly onto the Spin Cartridge.


3. Centrifuge at 3,300 × g in a microcentrifuge for 1 minute. Remove the collection tube and discard the flow-through.


4. Place the Spin Cartridge in the same collection tube and add 600 µl of Wash Buffer (prepared with ethanol) as described on page 14) to the column.


5. Centrifuge at maximum speed for 30 seconds. Remove the collection tube and discard the flow-through.


6. Place the Spin Cartridge in the same collection tube and centrifuge at maximum speed for 30 seconds to remove any residual Wash Buffer. Remove the collection tube and discard.


7. Place the Spin Cartridge onto a new amber collection tube (supplied in the kit).


8. Add 20 µl of DEPC-treated water to the center of the Spin Cartridge and incubate at room temperature for 1 minute.


9. Centrifuge at maximum speed for 1 minute to collect the purified cDNA. The eluate contains your purified labeled cDNA.

• Vortex mixer
• Microcentrifuge
• Water bath, incubator, or thermal cycler
• Aerosol resistant RNase-free pipette tips
• 0.5-ml or 1.5-ml RNase-free microcentrifuge tubes
• 0.1 N NaOH
• 0.1 N HCl
• 100% Isopropanol
• 100% Ethanol

1. Mix and briefly centrifuge each component before use.


2. In a 1.5-ml or 0.5-ml RNase-free tube, add the following:
TOP
 
Component                                                                                Volume
5–20 µg total RNA                                                                           X µl
Anchored oligo(dT)20 primer (2.5 µg/µl)                                         2 µl
DEPC-treated water                                                                  to 15 µl
 

3. Incubate tube at 70°C for 10 minutes, and then place on ice for at least 1 minute.


4. Add the following to the tube on ice:
    
   Component                                                                                                                                  Volume
   5X First-Strand buffer                                                                                                                        6 µl
   0.1 M DTT                                                                                                                                           3 µl
   10X Nucleotide Mix with Alexa Fluor® 555-aha-dUTP or
   10X Nucleotide Mix with Alexa Fluor® 647-aha-dUTP                                                                      3 µl
   RNaseOUT™ (40 U/µl)                                                                                                                       1 µl
   SuperScript™ III RT (400 U/µl)                                                                                                           2 µl
   Final Volume                                                                                                                                     30 µl
    
   


5. Mix gently and collect the contents of each tube by brief centrifugation. Note:   After addition of the labeled nucleotides, be careful to minimize exposure of the tube to light.


6. Incubate tube at 46°C in the dark for 3 hours. Note:   A 2-hour incubation is sufficient for generating high-quality labeled cDNA with high levels of picomole incorporation; however, a 3-hour incubation will result in 10–20% greater incorporation of labeled nucleotides and more full-length cDNA.

1. Add 15 µl of 0.1 N NaOH to each reaction tube from Step 6, above. Mix thoroughly.


2. Incubate tube at 70°C for 30 minutes.


3. Add 15 µl of 0.1 N HCl to neutralize the pH and mix gently.

1. Add 700 µl of Binding Buffer (prepared with isopropanol) as described on page 19) to the reaction tube containing the labeled cDNA from Hydrolysis and Neutralization, Step 3.


2. Each Low-Elution Volume Spin Cartridge is preinserted into a collection tube. For multiple reactions, clearly label each collection tube, and then load the cDNA/Binding Buffer solution directly onto the Spin Cartridge.


3. Centrifuge at 3,300 × g in a microcentrifuge for 1 minute. Remove the collection tube and discard the flow-through.


4. Place the Spin Cartridge in the same collection tube and add 600 µl of Wash Buffer (prepared with ethanol) to the column.


5. Centrifuge at maximum speed for 30 seconds. Remove the collection tube and discard the flow-through.


6.  Place the Spin Cartridge in the same collection tube and centrifuge at maximum speed for 30 seconds to remove any residual Wash Buffer. Remove the collection tube and discard.


7. Place the Spin Cartridge onto a new amber collection tube (supplied in the kit) 


8. Add 20 µl of DEPC-treated water to the center of the Spin Cartridge and incubate at room temperature for 1 minute.


9. Centrifuge at maximum speed for 1 minute to collect the purified labeled cDNA. The eluate contains your purified labeled cDNA.

Introduction

You can use UV/visible spectroscopy scanning to measure the amount of labeled cDNA and dye incorporation. The expected amounts using the Control HeLa RNA provided in the kit are shown below.
 
Calculating the Results

To calculate the amount of labeled cDNA using a UV/visible spectrophotometer:
 

1. Transfer a volume of purified, labeled cDNA to a clean cuvette. Use an appropriate volume for your spectrophotometer. Add DEPC-treated water to the cDNA if you need to increase the volume of the eluate for your spectrophotometer.
 
Note:   


2. Blank the spectrophotometer using DEPC-treated water, and then scan the sample at 240–800 nm. Wash each cuvette thoroughly between samples.


3. Calculate the yield of cDNA using the following formula:
    
   cDNA (ng) = (A₂₆₀–A₃₂₀) × 37 ng/µl × volume in µl


4. Calculate the amount of fluorescent dye using the following formulas:
    
   Alexa Fluor® 555 (pmole) = (A₅₅₅–A₆₅₀)/0.15 × volume in µl
   Alexa Fluor® 647 (pmole) = (A₆₅₀–A₇₅₀)/0.24 × volume in µl


5. Calculate the base-to-dye ratio using the following formulas:
    
   Base/dye ratio for Alexa Fluor® 555 =
   [(A₂₆₀ – A₃₂₀) – [(A₅₅₅– A₆₅₀) × 0.04]] × 150,000/(A₅₅₅ – A₆₅₀) × 8,919
    
   Base/dye ratio for Alexa Fluor® 647 =
   [(A₂₆₀ – A₃₂₀) – [(A₆₅₀ – A₇₅₀) × 0]] × 239,000/(A₆₅₀ – A₇₅₀) × 8,919

 
The number of dye molecules per 100 bases is calculated using the formula:
100/(base/dye ratio)
 
Expected Amounts Using Control DNA

If you prepared a control reaction using 10 µg of Control HeLa RNA as starting material, the following amounts are expected.
 
Indirect Labeling:
 

 Labeled cDNA                 Incorporated Dye                    Dyes Molecules/100 Bases
 ≥ 250 ng                             ≥ 24 pmole                                             ≥ 2.50
 
Direct Labeling:
 
Labeled cDNA             Incorporated Labeled Nuc.           Dyes Molecules/100 Bases
    ≥ 400 ng                             ≥ 30 pmole                                              ≥ 1.0
 
If you do not obtain these amounts, see Troubleshooting.
 
Because of the high purity of the cDNA from the Low-Elution Volume Spin Cartridges, the yield and picomole dye incorporation calculations will be more accurate than with other purification methods. 




In the 1.2% E-Gel above, Lanes 1 and 2 contain Alexa Fluor® 555-labeled cDNA purified using the Low-Elution Volume Spin Cartridges, and Lanes 3 and 4 contain Alexa Fluor® 555-labeled cDNA purified using columns from another manufacturer. The labeled cDNA appears as smear from 500–5,000 bp.

The large band at the bottom of Lanes 3 and 4 is unincorporated dye that was not removed by the other manufacturer’s purification column. Such material would be included in the picomole dye incorporation calculations, resulting in an incorporation level that is higher than theoretically possible.

• Make sure that buffers do not air dry on the slides between or after washes.
• Immediately centrifuge or use clean, filtered, compressed air to dry the slides after pre-hybridization and washing.
• Never use powdered gloves. Handle slides with forceps if possible, especially during the final wash step.

• Microarray—PCR product or oligonucleotide probes printed on Corning® GAPS, GAPS II, or UltraGAPS™ Slides
• Slide box, or clean, dry slide mailer(s)
• Hybridization chamber (e.g., Corning® Hybridization Chamber, catalog # 2551 or # 40080)
• Raised-edge coverslips (e.g., LifterSlips™, Erie Scientific, catalog # 22x50I-2-4711 or 22x60I-2-4861)
• Hybridization blocking DNA (e.g., Human Cot-1 DNA®, Mouse Cot-1 DNA®, or Salmon-Sperm DNA Solution)   Digital microarray scanner (e.g., the GenePix® 4000B from Molecular Devices) and associated software
• Slide rack
• Wash containers for individual slides (e.g., Coplin jars)
• Wash tanks, capable of completely submerging a slide rack
• Vortex mixer
• Orbital shaker
• Microcentrifuge
• Heat block (95°C)
• Water bath (42°C)
• Temperature-controlled incubator (42°C)
• Tabletop centrifuge with a microtiter plate rotor adapter capable of holding a slide rack or slide holder or filtered, compressed air
• Lint-free laboratory wipes
• Squirt Bottle
• Aerosol-resistant pipette tips
• 1.5-ml RNase-free microcentrifuge tubes

1. Pre-heat the Pre-Hybridization Solution (prepared with Buffer WB and formamide as described)  on page 25) to 42°C. Place the printed slides in a polypropylene slide mailer, and fill the mailer with enough pre-heated Pre-Hybridization Solution to completely submerge the slides.


2. Close the slide mailer, and invert gently 3–5 times.


3. Incubate the slide mailer at 42°C for 30 minutes. During the incubation, fill the wash containers as described in the next step, and you can begin preparing the hybridization mixture as described below.


4. Fill 5–6 wash containers (e.g., Coplin jars) with de-ionized water at room temperature (15–25°C). Process each slide individually in steps 5–8.
 
 Note:   Replace the de-ionized water in the containers after processing five slides.

5. At the end of the 42°C incubation, remove a single slide from the slide mailer. Submerge it in the first wash container and agitate for 5 seconds.


6. Transfer the slide immediately to the next wash container and agitate for 5 seconds. To avoid background spots, do not allow the liquid to dry on the slide between washes.


7. Repeat Step 6 until you have washed the slide once in each of the 5–6 wash containers.


8. To dry the slide, use one of the following methods (there should be no residue from the Pre-Hybridization Solution remaining on the dried slide):
   
   
   • Prepare a centrifuge with a microtiter plate rotor adapter that will accept the slide rack containing the array slide. (Balance the opposing arm of the rotor with a slide rack containing an equivalent number of empty slides.) Quickly transfer the slide rack with the slide to the centrifuge, and immediately spin for 2–4 minutes at 600 × g to dry. Do not centrifuge at higher speeds, or the slide might break.
   • Dry the slide under a stream of clean, filtered, compressed air. Hold the slide with the barcode at the bottom, and direct the stream of air from top to bottom, perpendicular to the slide surface. Let the water flow off smoothly and avoid streaks.
   
   


9. Repeat steps 5–8 for each slide. After all the slides are washed and dried, proceed to Preparing the Hybridization Mixture.

1. Warm the Hybridization Solution (prepared with buffer and formamide as described) to 42°C before starting, and ensure that any precipitate is completely dissolved.


2. Prepare a hybridization mixture as follows:
 
Component                                                                                                                 Volume per slide
Hybridization Solution                                                                                                               42 µl
Hybridization Blocking DNA, 10 mg/ml (e.g., Human Cot-1 DNA®,
Mouse Cot-1 DNA®, Salmon-Sperm DNA)                                                                               1 µl
Labeled cDNA (for dual-color hybridizations, combine both
labeling reactions as specified above)                                                                               ~16/32 µl
De-ionized Water                                                                                                                   to 84.0 µl
 
 

3. Gently vortex and heat the hybridization mixture at 95°C for 5 minutes. Centrifuge the tube briefly to bring down any condensation.

1. Chomczynski, P., and Sacchi, N. (1987) Single Step Method of RNA Isolation by Acid Guanidinium Thiocyanate-Phenol-Chloroform Extraction. Anal. Biochem., 162, 156-159


2. De Risi, J., Penland, L., Brown, P. O., Bittner, M. L., Meltzer, P. S. R., M., Chen, Y., Su, Y. A., and Trent, J. M. (1996) Use of a cDNA microarray to analyse gene expression patterns in human cancer. Nature Genet., 14, 457-460


3. Eisen, M. B., and Brown, P. O. (1999) DNA arrays for analysis of gene expression. Methods Enzymol., 303, 179-205


4. Gerard, G. F., D'Alessio, J. M., Kotewicz, M. L., and Noon, M. C. (1986) Influence on stability in Escherichia coli of the carboxy-terminal structure of cloned Moloney murine leukemia virus reverse transcriptase. DNA, 5, 271-279


5. Kotewicz, M. L., D'Alessio, J. M., Driftmier, K. M., Blodgett, K. P., and Gerard, G. F. (1985) Cloning and overexpression of Moloney murine leukemia virus reverse transcriptase in Escherichia coli. Gene, 35, 249-258