3’end Expression Array

MessageAmp II-biotin Enhanced kit(#AM1791 from Ambion)

 

Incubators needed:

• 70°C: thermal cycler recommended

• 42°C: hybridization oven or air incubator recommended

 

	Reverse Transcription to Synthesize First Strand cDNA
1. Mix RNA and 1 μl T7 Oligo(dT) Primer, and adjust volume to 12 μl	Place a maximum volume of 10 μl of total RNA (1000 ng-5000ng recommended) into a nonstick, sterile, RNase-free, 0.5 ml tube. RNA must be in high quality water or TE Add 1 μl of T7 Oligo(dT) Primer. Add Nuclease-free Water to a final volume of 12 μl, vortex briefly to mix, then centrifuge to collect the mixture at the bottom of the tube.
2. Incubate 10 min at 70°C, then place on ice	a. Incubate 10 min at 70°C in a thermal cycler. b. Centrifuge samples briefly (~5 sec) to collect them at the bottom of the tube. Place the mixtures on ice.
3. Add 8 μl of Reverse Transcription Master Mix and place at 42°C	At room temp, prepare Reverse Transcription Master Mix We provide a master mix calculator on our website to calculate reagent amounts: www.ambion.com/tools/ma2biotin   At room temp, assemble the Reverse Transcription Master Mix in the order shown: Reverse Transcription Master Mix (for a single 20 μl reaction) Amount Component 2 μl 10X First Strand Buffer 4 μl dNTP Mix 1 μl RNase Inhibitor 1 μl ArrayScript   b. Mix well by gently vortexing. Centrifuge briefly (~5 sec) to collect the Reverse Transcription Master Mix at the bottom of the tube and place on ice. c. Transfer 8 μl of Reverse Transcription Master Mix to each RNA sample. Mix thoroughly by pipetting up and down 2–3 times, then flicking the tube 3–4 times, and centrifuge briefly to collect the reaction in the bottom of the tube. d. Place the samples in a 42°C incubator.
4. Incubate for 2 hr at 42°C	Incubate reactions for 2 hr at 42°C (hybridization oven or air incubator is recommended). After the incubation, centrifuge briefly (~5 sec) to collect the reaction at the bottom of the tube. Place the tubes on ice and immediately proceed to second strand cDNA synthesis (below).

 

Incubator needed:

• 16°C: thermal cycler recommended

 

	D. Second Strand cDNA Synthesis
1. Add 80 μl Second Strand Master Mix to each sample	On ice, prepare a Second Strand Master   Assemble the Second Strand Master Mix on ice in the order shown: Second Strand Master Mix (for a single 100 μl reaction) Amount Component 63 μl Nuclease-free Water 10 μl 10X Second Strand Buffer 4 μl dNTP Mix 2 μl DNA Polymerase 1 μl RNase H   b. Mix well by gently vortexing. Centrifuge briefly (~5 sec) to collect the Second Strand Master Mix at the bottom of the tube and place on ice. c. Transfer 80 μl of Second Strand Master Mix to each sample. Mix thoroughly by pipetting up and down 2–3 times, then flicking the tube 3–4 times, and centrifuge briefly to collect the reaction in the bottom of the tube. d. Place the tubes in a 16°C thermal cycler. It is important to cool the thermal cycler block to 16°C before adding the reaction tubes because subjecting the reactions to temperatures >16°C will compromise aRNA yield.
2. Incubate for 2 hr at 16°C	Incubate 2 hr in a 16°C thermal cycler.    If the lid temperature cannot be adjusted to match the 16°C block temperature, cover the reactions with the heated lid turned off, or if the lid cannot be turned off—do not cover the tubes with it. (Do not use a water bath or a heat block in a 4°C refrigerator for this incubation because the temperature will fluctuate too much.) NOTE You may want to preheat the Nuclease-free Water, for use in step II.E.4, during this incubation.
3. Place reactions on ice briefly or freeze immediately	After the 2 hr incubation at 16°C, place the reactions on ice and proceed to section E. cDNA Purification (below), or immediately freeze reactions at –20°C. Do not leave the reactions on ice for more than 1 hr. STOPPING POINT This is a potential overnight stopping point (at –20°C), but it is better to complete the cDNA purification (next section) before stopping.

 

E.Cleanup of Double-Stranded DNA

Genechip sample cleanup module (#900371 from affymetrix)

 

Before starting,

• add 24 mL of Ethanol (100%) to the cDNA Wash Buffer supplied in the GeneChip Sample Cleanup Module.
• Preheat H2O(50-60°C)

 

 

1. Add 600 μL of cDNA Binding Buffer, and vortex for 3 seconds.
2. Apply 350ul of sample to a cDNA Spin Column sitting in a 2 mL Collection Tube.
3. Spin at ≥ 8,000 x g for 30sec. Discard the flow-through.
4. reload spin column with the remaining mixture(~350ul) and Spin at ≥ 8,000 x g for 30sec. Discard the flow-through and Collection tube.
5. Transfer the cDNA Cleanup Spin Column to a new 2 mL Collection Tube and add 650 μL of cDNA Wash Buffer to the column. Spin at ≥ 8,000 x g for 1 minute and discard the flow-through.
6. spin at ≤ 25,000 x g for 5 minutes with the caps open. Discard the flow-through, and place the column in a 1.5 mL collection tube.
7. Pipet 15 μL of the preheated H2O and incubate at room temperature for 1 minute. Then, spin at ≤ 25,000 x g for 1 minute.
8. Repeat the elution step by pipetting another 10 μL of the cDNA Elution Buffer and incubate at room temperature for 1 minute. Then, spin at ≤ 25,000 x g for 1 minute.
9. The total volume of the eluted Single-Stranded DNA is ~23 μL total.

 

 

F. In vitro Transcription to synthesize Biotin-labeled aRNA

1. Add 20 μl IVT Master Mix to each sample

At Room temperature, On ice, prepare a IVT Master Mix   Assemble the IVT Master Mix in the order shown:   Amount Component 20ul Double stranded cDNA IVT master mix for single reaction 12 μl  biotin-NTP Mix 4 μl    T7 10X reaction Buffer 4 μl    T7 enzyme mix   b. Mix well by gently vortexing. Centrifuge briefly (~5 sec) to collect the IVT Master Mix at the bottom of the tube and place on ice. c. Transfer 20 μl of IVT  Master Mix to each sample. Mix thoroughly by pipetting up and down 2–3 times, then flicking the tube 3–4 times, and centrifuge briefly to collect the reaction in the bottom of the tube. d. Place the tubes in a 37°C thermal cycler for 10hr then place 4°C overnight

G.Cleanup of Double-Stranded DNA

Genechip sample cleanup module (#900371 from affymetrix)

 

Before starting,

• add 20 mL of Ethanol (100%) to the cRNA Wash Buffer supplied in the GeneChip Sample Cleanup Module.
• Preheat H2O(50-60°C)

 

1.       Add 60 μL of RNase-free water to each IVT reaction to a final volume of 100 μL.

2.       Add 350μL of cRNA Binding Buffer, and vortex for 3 seconds.

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

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

5.       Centrifuge for 30 seconds at ≥ 8,000 x g. Discard the flow-through.

6.       reload sample again(~7000ul) and Spin at ≥ 8,000 x g for 30sec. Discard the flow-through and Collection tube.

7.       Transfer the cRNA Cleanup Spin Column to a new 2 mL Collection Tube and add 500 μL of cRNA Wash Buffer to the column. Spin at ≥ 8,000 x g for 1 minute and discard the flow-through.

8.       add 500 μL of 80%ETOH to the column. Spin at ≥ 8,000 x g for 1 minute and discard the flow-through.

9.       spin at ≤ 25,000 x g for 5 minutes with the caps open. Discard the flow-through, and place the column in a 1.5 mL collection tube

10.   Pipet 20 μL of the preheated H2O and incubate at room temperature for 3 minute. Then, spin at ≤ 25,000 x g for 1 minute.

11.   Second Pipet 20 μL (total 40ul)of the preheated H2O and incubate at room temperature for 3 minute. Then, spin at ≤ 25,000 x g for 1 minute.

 

 Measure cRNA conceturation.

 

Eukaryotic Target Hybridization

Please refer to the table below for the necessary amount of cRNA required for appropriate

probe array format. These recipes take into account that it is necessary to make extra

hybridization cocktail due to a small loss of volume (10-20 μL) during each hybridization.

1. Mix the following for each target, scaling up volumes for hybridization to multiple

probe arrays.

Table 2.2.1

Hybridization Cocktail for Single Probe Array*

Component	49 Format (Standard) /64 Format Array	100 Format (Midi) Array	169 Format (Mini) Array /400 Format (Micro) Array	Final Concentration
Fragmented cRNA **	15 μg	10 μg	5 μg	0.05 μg/μL
Control Oligonucleotide B2 (3 nM)	5 μL	3.3 μL	1.7 μL	50 pM
20X Eukaryotic hybridization Controls (bioB, bioC, bioD, cre)	15 μL	10 μL	5 μL	1.5, 5, 25, and 100 pM respectively
Herring Sperm DNA (10 mg/mL)	3 μL	2 μL	1 μL	0.1 mg/mL
BSA (50 mg/mL)	3 μL	2 μL	1 μL	0.5 mg/mL
2X Hybridization Buffer	150 μL	100 μL	50 μL	1X
DMSO***	30 μL	20 μL	10 μL	10%
H2O μL	to final volume of 300	to final volume of 200 μL	to final volume of 100 μL
Final volume	300 μL	200 μL	100 μL

*Please refer to specific probe array package insert for information on array format.

**Please see Section 2, Chapter 1, for amount of adjusted fragmented cRNA to use when starting from total RNA.

*** Note that the addition of DMSO is different from previous recommendations. Follow this protocol for best results on arrays when

using the GeneChip IVT Labeling Kit.

2. Equilibrate probe array to room temperature immediately before use.

3. Heat the hybridization cocktail to 99°C for 5 minutes in a heat block.

4. Meanwhile, wet the array by filling it through one of the septa (see Figure 2.2.1 for location of the probe array septa) with appropriate volume of 1X Hybridization Buffer using a micropipettor and appropriate tips (Table 2.2.2).

5. Incubate the probe array filled with 1X Hybridization Buffer at 45°C for 10 minutes with rotation.

6. Transfer the hybridization cocktail that has been heated at 99°C, in step 3, to a 45°C heat block for 5 minutes.

7. Spin hybridization cocktail(s) at maximum speed in a microcentrifuge for 5 minutes to remove any insoluble material from the hybridization mixture.

8. Remove the buffer solution from the probe array cartridge and fill with appropriate volume (Table 2.2.2) of the clarified hybridization cocktail, avoiding any insoluble matter at the bottom of the tube.

9. Place probe array into the Hybridization Oven, set to 45°C.

Avoid stress to the motor; load probe arrays in a balanced configuration around the axis. Rotate at 60 rpm.

10. Hybridize for 16 hours.

During the latter part of the 16-hour hybridization, proceed to Section 2, Chapter 3 to prepare reagents required immediately after completion of hybridization.

 

Preparing the Staining Reagents

1. Prepare the following stain and wash solutions Do not freeze SAPE. Always prepare the SAPE stain solution fresh, on the day of use.

 

SAPE Solution Mix - Vial 1

Components	Volume
2X Stain Buffer	600.0 μL
50 mg/mL BSA	48.0 μL
1 mg/mL Streptavidin Phycoerythrin(SAPE)	12.0 μL
Nuclease-free H20	540.0 ulL
Total Volume	1200 μL

 

 

Antibody Solution Mix - Vial 2

Components	Volume
2X Stain Buffer	300.0 μL
50 mg/mL BSA	24.0 μL
10 mg/mL Normal Goat IgG	6.0 μL
0.5 mg/mL Anti-streptavidin Antibody, biotinylated	3.6μL
Nuclease-free H20	266.4ulL
Total Volume	600 μL

 

Washing and Staining the Probe Array

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

2. In the Protocol drop-down list, select the appropriate antibody amplification protocol to control the washing and staining of the probe array format being used: Table 2.3.3 and Table 2.3.4.

3. Choose Run in the Fluidics Station dialog box to begin the washing and staining.

Follow the instructions in the LCD window on the fluidics station. If you are unfamiliar with inserting and removing probe arrays from the fluidics station modules, please refer to the appropriate Fluidics Station User’s Guide or Quick

Reference Card (P/N 08-0093 for the FS-450/250 fluidics stations).

4. Insert the appropriate probe array into the designated module of the fluidics station while the cartridge lever is in the down, or eject, position. When finished, verify that the cartridge lever is returned to the up, or engaged, position.

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

6. If prompted to “Load Vials 1-2-3,” place the three experiment sample vials (the microcentrifuge vials) into the sample holders 1, 2, and 3 on the fluidics station.

■ Place one vial containing 600 μL of streptavidin phycoerythrin (SAPE) solution in sample holder 1.

■ Place one vial containing 600 μL of anti-streptavidin biotinylated antibody solution in sample holder 2.

■ Place one vial containing 600 μL of streptavidin phycoerythrin (SAPE) solution in sample holder 3.

■ 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 the LCD window display the status of the washing and staining as they progress.

7. At the end of the run, or at the appropriate prompt, remove the microcentrifuge vials and replace with three empty microcentrifuge vials.

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

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

■ If bubbles are present, proceed to Table 2.3.6.

■ If the probe array has no large bubbles, it is ready to scan on the GeneArray® Scanner or the GeneChip® Scanner 3000. Pull up on the cartridge lever to engage washblock and proceed to Probe Array Scan on page 2.3.15.

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

 

Table 2.3.6

If Bubbles are Present Return the probe array to the probe array holder. Engage the washblock by gently pushing up

on the cartridge lever to the engaged, or closed, position.

The fluidics station will drain the probe array and then fill it with a fresh volume of the last wash buffer used. When it is finished, the LCD window will display EJECT CARTRIDGE. Again, remove the probe array and inspect it for bubbles. If no bubbles are present, it is ready to scan. Proceed to Probe Array Scan on page 2.3.15.

If several attempts to fill the probe array without bubbles are unsuccessful, the array should be filled with Wash Buffer A (non-stringent buffer) manually, using a micropipette. Excessive washing will result in a loss of signal intensity.