Whole Transcript (WT) Sense Target Protocol for Exon ST array

Suggested Workflow

Day 1

• Complete Chapter 2: rRNA Reduction – ~1.5 hours (for GeneChip Exon 1.0 ST Arrays)

• Complete Chapter 3 or 4, Procedures A – C: First Cycle: Synthesis of cDNA – ~3.5 hours

• Start Chapter 3, Procedure D: First Cycle: Synthesis of cRNA – 16 hours – Start on

Day 1, finish on Day 2

Day 2

• Complete Chapter 3, Procedures D – I: Second Cycle: Synthesis of first-strand DNA,

fragmentation , and labeling – 8 hours

• Start Chapter 5: Hybridization – 17 hours – Start on Day 2, finish on Day 3

Day 3

• Chapter 6: Array Washing, Staining, and Scanning – 2 hours

 

rRNA Reduction and Preparation of Total RNA With Diluted Poly -A RNA Controls

 

Procedure A: Preparation of Dilutions of Poly-A RNA Controls

This Procedure requires the use of the GeneChip® Poly-A RNA Control Kit.

 

The following protocol requires a minimum of 1 μg of total RNA as starting material,and theconcentration should not fall below 0.31 μg/μL. In other words, the 1 μg of totalRNA should be suspended in a maximum of 3.2 μL of solution in volume. One to 2 μgof total RNA may be used, but the RNA concentration must be high enough because themaximum volume of total RNA is 3.2 μL.

 

1.      Add 2ul of Poly-A control Stock to 38ul Poly-a dilution buffer to make the First Dilution

( 1:20 dilution). Mix well and spin down.

2.      Add 2ul of First Dilution to 98ul Poly-A dilution buffer to make Second Dilution (1:50 dilution).

Mix well and spin down.

3.      When using 1 μg of total RNA as starting material, Add 2ul of Second Dilution to 98ul Poly-A dilution buffer to make Third Dilution (1:50 dilution).

When using 2 μg of total RNA as starting material, Add 2ul of Second Dilution to 98ul Poly-A dilution buffer to make Third Dilution (1:25 dilution).

Mix and spin to collect the solution at the bottom of the tube. Add 2 μL of theappropriate Third Dilution to 1 μg or 2 μg of total RNA to make up the Total RNA/Poly-A RNA Controls Mix as described in Table 2.1.

 

Starting Amount of Total RNA	Serial Dilutions	Volume into Sample
	First                    Second            Third
1ug	1:20                              1:50                          1:50	2 ul
2 ug	1:20                              1:50                          1:25	2 ul

 Table 2.1 Poly-A RNA Control Stock Serial Dilutions

 

4.      Store First Dilution, Second Dilution and Third Dilution  at -80ºC.

Procedure B: Preparation of Hybridization Buffer with Betaine

This Procedure requires the use of the RiboMinus Human/Mouse TranscriptomeIsolation Kit from Invitrogen.

1. Prepare the buffer by mixing these components as listed in Table 2.2. Keep tube at room temperature.

	Starting Total RNA Amount
	1 ug	2 ug
Component	Volume for 1 Rxn	Volume for 1 Rxn
Betaine, 5M	54 ul	84 ul
Invitrogen Hybidization Buffer	126 ul	196 ul
Total Volume	180 ul	280

Table 2.2 Hybridization Buffer with Betaine

  * A 30 ul overfill is included in the Total Volume, sufficient for completing the RivoMiuns pricedure for a single total RNA sample.

 

Procedure C: RiboMinus Probe Hybridization

This Procedure requires the use of the RiboMinus Human/Mouse TranscriptomeIsolation Kit that needs to be obtained directly from Invitrogen.

1. mix the following components in Table 2.3 depend on total RNA amount. Keep the tubes on ice.

 

Total RNA Amount	1 ug	1 ug	2 ug
RNA Concentration Range	1 ug/ul	0.31 ug/ul to 1 ug/ul	≥0.62 ug/ul
RiboMinus Probe Hybridization
Total RNA/Poly-a Controls Mix (from Procedure A)	3.0 ul	Up to 5.2 ul	Up to 5.2 ul
RiboMinus Probe (100 pmol/uL)	0.8 ul	0.8 ul	1.6 ul
Hybrodization Buffer with Betaine (from Procedure B)	20 ul	30 ul	40 ul
Total Hybridization Volume	23.8 ul	36.0 ul	46.8 ul

 Table 2.3 RiboMinum Reaction and Wash Volumes

2. Flick the tube gently to mix, spin briefly and incubate at 70°C for 5 minutes in athermal cycler.

3. Quench the reaction immediately by placing the tube on ice while preparing the magnetic beads

Procedure D: Preparation of Beads

This Procedure requires the use of the RiboMinus Human/Mouse TranscriptomeIsolation Kit that needs to be obtained directly from Invitrogen.

 

NOTE: Procedures D and E require the use of a 37°C heat block and a 50°Cheat block. Pre-heat the heat blocks prior to initiating these steps.

 

1. Completely re-suspend   magnetic beads by flicking it  

2. Pipet 50 μL (100 μL) of beads suspension into a 1.5 mL non-stick RNase-free tube.

Steps 3 to 6 are done at room temperature.

3. Briefly spin and place the tube with the beads suspension on the magnetic stand for

~1 minute. With the tube remaining in the stand, gently aspirate and discard thesupernatant.

4. 1st Wash :

   A. Add 50 μL (100 μL) of RNase-free water to the beads and re-suspend them byflicking the tube.

NOTE: If the beads are still attached to the wall of the tube, put thetube back into the magnetic stand and rotate the tube with quickmotion until the beads are in suspension

  B. Spin briefly. Place the tube on the magnetic stand for ~1 minute. With the tuberemaining in the stand, gently aspirate and discard the supernatant.

5. 2nd Wash : Add 50 μL (100 μL) of RNase-free water to the beads and re-suspendthem by flicking the tube. Place the tube on the magnetic stand for ~1 minute. Withthe tube remaining in the stand, gently aspirate and discard the supernatant.

6. 3rd Wash : Add 50 μL (100 μL) of the Hybridization Buffer with Betaine (fromProcedure B) to the beads and re-suspend them by flicking the tube. Spin briefly.Place the tube on the magnetic stand for ~1 minute. With the tube remaining in thestand, gently aspirate and discard the supernatant.

7. Re-suspend the beads in Hybridization Buffer with Betaine. Keep them at 37°C in aheating block for 1 to 2 minutes. Refer to Table 2.3 for appropriate volume.

 

NOTE: If there is a large number of samples to be processed at the sametime the beads can be prepared in a large batch. This can be done with up toa total of 10 samples, Following the3rd Wash (Procedure D, Step 6), the resuspended beads can then bealiquoted to individual tubes as described in Procedure D, Step 7 beforeproceeding to rRNA Reduction for individual samples.

Procedure E: rRNA Reduction

Two heating blocks are required: one at 37°C, and the other at 50°C. Use the volumesbetween parenthesis if the starting amount of total RNA is 2 μg.

 

1. Transfer the ice-cooled hybridized sample prepared in Procedure C to the beadsprepared in Procedure D, mix well, and briefly spin.

2. Incubate at 37°C for 10 minutes in a heating block. After5 minutes of incubation, gently flick-mix

3. Briefly spin and place the tube in the magnetic stand for 1minutes to obtain therRNA -probe pellet.

NOTE: The supernatant contains the rRNA-Reduced Total RNA/Poly-A RNAControls Mix.

4. With the tube in the magnetic stand, transfer the supernatant to a 1.5 mL non-stickRNase -free tube and leave on ice.

5. Wash the beads by re-suspending them in 50 μL (50 μL) of Hybridization Bufferwith Betaine and incubate at 50ºC for 5 minutes.

6. Place the tube in the magnetic stand for 1 to 2 minutes, transfer the supernatant, andcombine with the supernatant in the tube from Procedure E, Step 4. The total volumeof the rRNA-reduced sample is approximately 100 μL (150 μL).

 

Procedure F: Concentration

This Procedure requires the use of the GeneChip® IVT cRNA Cleanup Kit.

1. Add 350 μL (525 μL) of cRNA Binding Buffer to each rRNA-reduced sample fromProcedure E and vortex for 3 seconds.

2. Add 250 μL (375 μL) of 100% ethanol to each reaction and flick the tube to mix.

3. Transfer the sample to the IVT cRNA Cleanup Spin Column sitting in a 2 mL Collection Tube.

4. Centrifuge for 15 seconds at ≥8,000 x g. Discard the flow-through.

5. Transfer the IVT cRNA Cleanup Spin Column to a new 2 mL Collection Tube.

Add 500 μL of cRNA Wash Buffer and centrifuge for 15 seconds at ≥8,000 x g.Discard.

6. Wash again with 500 μL of 80% (v/v) ethanol. Centrifuge for 15 seconds at≥8,000 x g and discard the flow-through.

7. Open column cap and spin at ≤25,000 x g (maximum speed) for 5 minutes with thecap left open.

8. Transfer the IVT cRNA Cleanup Spin Column to a new 1.5 mL Collection Tube andadd 11 μL of RNase-free Water. Spin at ≤25,000 x g(maximum speed) for 1 minute.

9. The eluted rRNA-Reduced Total RNA/Poly-A RNA Controls Mix is ~9.8 μL.

Keep sample on ice. Proceed with Procedure G (Analysis with Bioanalyzer) andChapter 3, Procedure A.

 

IMPORTANT: If the starting amount of total RNA is 2 μg, the totalsupernatant volume will exceed the capacity of the column. Apply~700 μL, centrifuge for 15 seconds at ≥8,000 x g. Discard the flowthrough.Place the column back in to 2 mL collection tube and apply theremaining sample (~350 μL). Continue to Step 6.

NOTE: Store eluted rRNA-reduced total RNA at –80°C if not proceedingimmediately to Procedure G (Analysis with Bioanalyzer) and Chapter 3,Procedure A.

Chapter3

1 μg TOTAL RNA TARGET LABELING PROTOCOL

 

Procedure A: Preparation of rRNA-Reduced Total RNA/Poly-A RNAControls/T7-(N)6 Primers Mix

This Procedure requires the use of the GeneChip® WT cDNA Synthesis Kit.

1. Dilute the T7-(N)6Primers, 2.5 μg/μL stock 1:5 with RNase-free water to make up a500 ng/μL working solution. Keep on ice. Prepare fresh every time.

2. Mix the diluted T7-(N)6Primers with the Concentrated rRNA-Reduced Sample fromChapter 2, Procedure F as listed in Table 3.1, using 0.2 mL strip tubes on ice.

Component	Volumn in 1 Rxn
rRNA-Reduced Total RNA/Poly-A RNA Control Mix	4 uL
Diluted T7-(N)6 Primers, 500 ng/uL	1 uL
Total Volume	5 uL

Table 3.1 First-cycle, Preparation of rRNA-reduced Total RNA/Poly-A RNA Controls/T7-(N)₆

3. Flick the tube to mix, spin down the tube, and incubate at:

• 70°C for 5 minutes

• 4°C for at least 2 minutes

4. Spin down and place on ice for use in Procedure B.

 

Procedure B: First-Cycle, First-Strand cDNA Synthesis

This Procedure requires the use of the GeneChip® WT cDNA Synthesis Kit.

1. Prepare Master Mix as shown in Table 3.2..

Component	Volume in 1 Rxn
5x 1st Strand Buffer	2 uL
DTT, 0.1M	1 uL
dNTP Mix, 10mM	0.5 uL
RNase Inhibitor	0.5 uL
SuperScript II	1 uL
Total Volume	5 uL

  Table 3.2 First-Cycle, First-Strand Master Mix

 

2. Add 5 μL of Master Mix to the tube containing theoncentrated rRNA-Reduced Total RNA/Poly-A RNA Controls/ T7-(N)6Primers

Mix from Procedure A, flick-mix, and spin-down. The total reaction volume is10 μL.

3. Incubate the reaction at:

• 25°C for 10 minutes

• 42°C for 60 minutes

• 70°C for 10 minutes

4. Cool the reaction to 4°C for at least 2 minutes before immediately continuing to theFirst-Cycle, Second-Strand cDNA Synthesis.

NOTE: Keeping the reaction at 4°C longer than 10 minutes may result inreduced cRNA yields.

 

 

 

Procedure C: First-Cycle, Second-Strand cDNA Synthesis

This Procedure requires the use of the GeneChip® WT cDNA Synthesis Kit.

1. Make a fresh dilution of 17.5 mM MgCl2 each time. Mix 2 μL of 1M MgCl2 with

112 μL of RNase-free water.

2. Prepare Master Mix as described in Table 3.3.

Component	Volume in 1 Rxn
RNase-free Water	4.8 uL
MgCl2, 17.5 mM	4.0 uL
dNTP Mix, 10 mM	0.4 uL
DNA Polymerase I	0.6 uL
RNase H	0.2 uL
Total Volume	10.0 uL

  Table 3.3 First-Cycle, Second-Strand Master Mix

 

3. Add 10 μL Master Mix to the reaction tube fromthe First-Strand cDNA Synthesis Reaction in Procedure B for a total reaction volumeof 20 μL. Flick or gently vortex the tubes and spin down.

4. Incubate the reaction in a thermal cycler at:

• 16°C for 120 minutes without heated lid

• 75°C for 10 minutes with heated lid

5. Cool the sample for at least 2 minutes at 4°C before immediately proceeding to thenext Procedure: First-Cycle, cRNA Synthesis and Cleanup.

NOTE: Keeping the reaction at 4°C longer than 10 minutes may result inreduced cRNA yields. 

 

Procedure D: First-Cycle, cRNA Synthesis and Cleanup

This Procedure requires the use of the GeneChip® WT cDNA Amplification Kit and the

GeneChip ® Sample Cleanup Module.

1. make the IVT Master Mix at room temperature as listed inTable 3.4.

Component	Volume in 1 Rxn
10x IVT Buffer	5.0 uL
IVT NTP Mix	20.0 uL
IVT Enzyme Mix	5.0 uL
Total Volume	30.0 uL

Table 3.4 First-Cycle, IVT Master Mix

2. Transfer 30 μL of the IVT Master Mix to each First-Cycle cDNA Synthesis Reactionsample from Procedure C to a final volume of 50 μL. Flick-mix the solution, andbriefly spin in a microfuge.

3. Incubate the reaction for 16 hours at 37°C.

4. Proceed to the cleanup procedure for cRNA using the cRNA Cleanup Spin Columnsfrom the GeneChip Sample Cleanup Module Store the sample at –80°C if not purifying the cRNA immediately.

 

6. Add 50 μL of RNase-free water to each IVT reaction to a final volume of 100 μL.

7. Add 350 μL of cRNA Binding Buffer to each sample and vortex for 3 seconds.

8. Add 250 μL of 100% ethanol to each reaction and flick-mix.

9. Apply the sample to the IVT cRNA Cleanup Spin Column sitting in a 2 mLCollection Tube.

10. Centrifuge for 15 seconds at ≥ 8,000 x g. Discard the flow-through.

11. Transfer the IVT cRNA Cleanup Spin Column to a new 2 mL Collection Tube.

Add 500 μL of cRNA Wash Buffer to column and centrifuge for 15 seconds at≥ 8,000 x g. Discard the flow-through.

12. Wash again with 500 μL of 80% (v/v) Ethanol. Centrifuge for 15 seconds at≥ 8,000 x g and discard the flow-through.

13. spin at ≤ 25,000 x g (maximum speed) for 5 minutes withthe caps open.

14. Transfer the IVT cRNA Cleanup Spin Column to a new 1.5 mL Collection Tube andadd 15 μL of RNase-free water directly to the membrane. Incubate at roomtemperature for 5 minutes. Spin at ≤ 25,000 x g (maximum speed) for 1 minute.

15. Elute a second time by pipetting the flow-through in the Collection Tube (~13.5 μL)back onto the Spin Column membrane. Place the Spin Column back into theCollection Tube and incubate at room temperature for 5 minutes.Spin at ≤ 25,000 x g (maximum speed) for 1 minute.

 

NOTE: Store eluted cRNA at –80°C if not proceeding immediately toProcedure E.

NOTE: If cRNA concentration is too low to obtain 8 to 10 μg of cRNA in 6.5 μL,cRNA can be concentrated using a SpeedVac.

 

Procedure E: Second-Cycle, First-Strand cDNA Synthesis

This Procedure requires the use of the GeneChip® WT cDNA Synthesis Kit.

1. Mix cRNA sample from Procedure D with the Random Primers, aslisted in Table 3.5 below.

Component	Volume in 1 Rxn
cRNA, 10 ug*	variable
Random Primers (3 ug/uL)	1.5 uL
RNase-free water	up to 8 uL
Total Volume	8.0 uL

 *For some samples that generate high yield in the second-cycle cDNA synthesis reaction, 8 ug may be used to obtain 5 ug of single-stranded DNA target.  The starting amount of cRNA varies depending on the type of tissue used.

 Table 3.5 Second-cycle, cRNA/Random Primers Mix

 

2. Flick-mix and spin down the tubes.

3. Incubate the Second-Cycle, cRNA/Random Primers Mix at:

• 70°C for 5 minutes

• 25°C for 5 minutes

4. Cool the samples at 4°C for at least 2 minutes.

5. In a separate tube,

prepare the Second-Cycle, Reverse Transcription Master Mix as described in Table 3.6. 

Component	Volume in 1 Rxn
5x 1st Strand Buffer	4.0 uL
DTT, 0.1M	2.0 uL
dNTP+dUTP, 10mM	1.25 uL
SuperScript II	4.75 uL
Total Volume	12.0 uL

  Table 3.6 Second-Cycle, First-Strand cDNA Synthesis Master Mix

 

6. Transfer 12 μL of Master Mix to theSecond-Cycle, cRNA/Random Primers Mix from Procedure E, Step 4 for a totalreaction volume of 20 μL. Mix thoroughly by gently flicking the tubes a few times

and centrifuge briefly.

7. Incubate the reactions at:

• 25°C for 10 minutes

• 42°C for 90 minutes

• 70°C for 10 minutes

• 4°C for at least 2 minutes

 

 

Procedure F: Hydrolysis of cRNA and Cleanup of Single-Stranded DNA

This Procedure requires the use of the GeneChip® WT cDNA Synthesis Kit and the

GeneChip ® Sample Cleanup Module.

1. Add 1 μL of RNase H to each of the samples and incubate at:

• 37°C for 45 minutes

• 95°C for 5 minutes

• 4°C for 2 minutes

 

4. Add 80 μL of RNase-free water to each sample, followed by 370 μL of cDNABinding Buffer, and vortex for 3 seconds.

5. Apply the entire sampleto a cDNA Spin Column sittingin a 2 mL Collection Tube.

6. Spin at ≥ 8,000 x g for 1 minute. Discard the flow-through.

7. Transfer the cDNA Cleanup Spin Column to a new 2 mL Collection Tube and add750 μL of cDNA Wash Buffer to the column. Spin at ≥ 8,000 x g for 1 minute anddiscard the flow-through.

8 spin at ≤ 25,000 x g for5 minutes with the caps open. Discard the flow-through, and place the column in a1.5 mL collection tube.

9. Pipet 15 μL of the cDNA Elution Buffer andincubate at room temperature for 1 minute. Then, spin at ≤ 25,000 x g for 1 minute.

10. Repeat the elution step by pipetting another 15 μL of the cDNA Elution Bufferand incubate at room temperature for 1 minute.Then, spin at ≤ 25,000 x g for 1 minute.

11. The total volume of the eluted Single-Stranded DNA is ~28 μL total.

 

to determine the yield

Each tube should have ≥ 5.5 μg of Single-Stranded DNA .

NOTE: Eluted single-stranded cDNA can be stored overnight at –20°C if notproceeding immediately to Procedure G.

 

Procedure G: Fragmentation of Single-Stranded DNA

This Procedure requires the use of the GeneChip® WT Terminal Labeling Kit.

1. Set up fragmentation reaction in 0.2 mL strip tubes using Table 3.7.

Component	Volume/Amount in 1 Rxn
Single-Strand DNA	5.5 ug
RNase-free Water	up to 31.2 uL
Total Volume	31.2 uL

Table 3.7 Fragmentation Master Mix

2. Prepare the Fragmentation Master Mix using Table 3.8.

Component	Volume in 1 Rxn
RNase-free Water	10 uL
10x cDNA Fragmentation Buffer	4.8 uL
UDG, 10 U/uL	1.0 uL
APE 1, 1,000 U/uL	1.0 uL
Total Volume	16.8 uL

  Table 3.8 Fragmentation Master Mix

3. Add 16.8 μL of the above Fragmentation Master Mix to the samples prepared inStep 1. Flick or gently vortex the tubes and spin down.

4. Incubate the reactions at:

• 37°C for 60 minutes

• 93°C for 2 minutes

• 4°C for at least 2 minutes

5. Flick-mix, spin down the tubes, and transfer 45 μL of the sample to a new 0.2 mLstrip tube.

6. If the samples are not labeled immediately, store the fragmented Single-StrandedDNA at –20°C.

 

Procedure H: Labeling of Fragmented Single-Stranded DNA

This Procedure requires the use of the GeneChip® WT Terminal Labeling Kit.

1. prepare a master mix using the 5X TdTBuffer, TdT and DNA Labeling reagent as below table.

   Component	Volume
   5x TdT Buffer	12 uL
   TdT	2 uL
   DNA Labeling Reagent, 5 mM	1 uL

2. aliquoting 15 μL into tubes containing the 45 μL of Fragmented Single-StrandedDNA .
3. flick-mix andspin them down.

Incubate the reactions at

• 37°C for 60 minutes

• 70°C for 10 minutes

• 4°C for at least 2 minutes

 

Chapter5 HYBRIDIZATION

 

ProcedureA : Hybridization , washing and staining

 

Hybridization

This Procedure requires the use of the GeneChip® Hybridization, Wash and Stain Kit.

Three heating blocks are required: one at 65°C, one at 99°C, and the third one at 45°C.

1. Prepare the Hybridization Cocktail in a 1.5 mL RNase-free microfuge tube as shown

in Table 5.1.

Table 5.1 Hybridization Cocktail

 

Component	Volume for One 49/64 Format Array	Volume for One 169 Format Array	Final Concentration
Fragmented and Labeled DNATarget (from Chapter 3)	~60.0* μL	27 μL	25 ng/μL
Control Oligonucleotide B2 (3 nM)	3.7 μL	1.7 μL	50 pM
20X Eukaryotic Hybridization Controls (bioB, bioC, bioD, cre)	11 μL	5 μL	1.5, 5, 25 and100 pM, respectively
2X Hybridization	Mix 110 μL	50 μL	1X
DMSO	15.4 μL	7 μL	7%
Nuclease-free Water	up to 220.0 μL	up to 100
Total Volume	220.0 μL	100 μL

* This volume is 58 μL if a portion of the sample was set aside for Gel-shift analysis.

2. Flick or gently vortex the tubes and spin down.

 

IMPORTANT: It is imperative that frozen stocks of 20X GeneChip®Eukaryotic Hybridization Controls are heated to 65°C for 5 minutes tocompletely resuspend the cRNA before aliquoting

 

 

 

3. Heat the Hybridization Cocktail at 99°C for 5 minutes. Cool to 45°C for 5 minutes,and centrifuge at maximum speed for 1 minute.

4. Equilibrate the GeneChip ST Array to room temperature immediately before use.Label the array with the name of the sample that will be hybridized.

5. Inject the appropriate amount (see Table 5.2) of the specific sample into the arraythrough one of the septa (see Figure 5.1 for location of the septa on the array).

6. Place array in 45°C hybridization oven, at 60 rpm, and incubate for 17 hours ± 1 hour. during the latter part of the array hybridization, commence preparation of thereagents required immediately after completion of hybridization.

 

Preparing the Staining Reagents

Prepare the following reagents. Volumes given are sufficient for one probe array.

1. Remove Stain Cocktail 1, Stain Cocktail 2, and Array Holding Buffer from the StainModule, Box 1 .

2. Gently tap the bottles to mix well.

3. Aliquot the following reagents:

A. 600 μL of Stain Cocktail 1 into a 1.5 mL amber microcentrifuge vial.

B. 600 μL of Stain Cocktail 2 into a 1.5 mL (clear) microcentrifuge vial.

C. 800 μL of Array Holding Buffer into a 1.5 mL (clear) microcentrifuge vial.

4. Spin down all vials to remove the presence of any air bubbles.

 

Washing and Staining the Probe Array on Fluidics Station 450

1. In the Fluidics Station dialog box on the workstation, select the correct experimentname from the drop-down Experiment list.The Probe Array Type appears automatically.

2. In the Protocol drop-down list, select FS450_0001 or FS450_0007 to control thewashing and staining steps. Refer to Table 6.3 for information on determining thecorrect Protocol for your array.

3. Choose Run in the Fluidics Station dialog box to begin the washing and staining.Follow the instructions on the LCD window on the fluidics station.If you are unfamiliar with inserting and removing probe arrays from the fluidicsstation modules, please refer to the appropriate Fluidics Station User’s Guide orQuick Reference Card (P/N 08-0093 for the FS-450/250 fluidics station).

4. Insert the appropriate probe array into the designated module of the fluidics stationwhile the cartridge lever is in the down, or EJECT position. When finished, verifythat the cartridge lever is returned to the up, or ENGAGE position.

5. Remove any microcentrifuge vial remaining in the sample holder of the fluidicsstation module(s) being used.

6. Follow the instructions on the LCD window on the fluidics station by placing thethree experiment sample vials (the microcentrifuge vials) into the sample holders 1,2, and 3 on the fluidics station.

A. Place one vial containing 600 μL Stain Cocktail 1 in sample holder 1.

B. Place one vial containing 600 μL Stain Cocktail 2 in sample holder 2.

C. Place one vial containing 800 μL of Array Holding Buffer in sample holder 3.

D. Press down on the needle lever to snap needles into position and to start the run.The run begins. The Fluidics Station dialog box at the workstation terminal and theLCD window display the status of the washing and staining as the protocol progresses.

7. When the protocol is complete, the LCD window displays the message EJECT &INSPECT CARTRIDGE.

8. Remove the probe arrays from the fluidics station modules by first pressing down thecartridge lever to the eject position.

9. Check the probe array window for large bubbles or air pockets.

• If the probe array has no large bubbles, it is ready to scan on the GeneChip®Scanner 3000 7G. Pull up on the cartridge lever to engage washblock and proceedto Scanning on page 45.

• If bubbles are present, do the following:

Return the probe array to the probe array holder. Follow instructions on the LCDwindow. Engage the washblock by gently pushing up on the cartridge lever to theengaged, or closed, position

The fluidics station will drain the probe array and then fill it with a fresh volume ofArray Holding Buffer. When it is finished, the LCD window will display EJECT &INSPECT CARTRIDGE. Again, remove the probe array and inspect it for bubbles.If no bubbles are present, it is ready to scan. Pull up on the lever to close thewashblock and proceed to Scanning on page 45.

If attempt to fill the probe array without bubbles is unsuccessful, the array should befilled manually with Array Holding Buffer using a micropipette with volumes listedin Table 6.1. Excessive washing will result in a loss of signal intensity.

10. If you do not scan the arrays right away, keep the probe arrays at 4°C and in the darkuntil ready for scanning.

11. If there are no more arrays to wash, shut down the fluidics station following theprocedure outlined in the section, Shutting Down the Fluidics Station on page 44.