Edge lab Agilent Microarray Labeling Protocol 07/03/2012

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Notes to remember:
- Samples and reagents take 2 hours to thaw.
- Vortex samples and quick-spin when thawed.
- Flick reagents and quick-spin when thawed.
- When finished with reagents, mark the number of samples that are used.
- Wipe tubes when removing them from a water bath and/or ice box to prevent
contamination. Considering how small the total volumes are, any small change in volume
will seriously screw you over!
- Cy3 is extremely photoreactive. When not using it, keep it covered under foil.
- Take two of each reagent so as to avoid scrambling in the chance you run out of reagents.
Considering that the reagents take 2 hours to thaw, it would take a really long time.
- These reagents have really small volumes, so every drop counts. Be very careful and
wipe off any moisture on the sides to avoid contamination!
- It would be a good idea to quantify before beginning, as well as after the cDNA step
(done using the nucleic acid tab under DNA) and after the cRNA step (done using the
microarray tab under RNA).
o With these last two quantification steps, remember to try and recover as much as
possible. The total volume of each sample is really small so every drop counts!
- If the yield is <1.65 g and the specific activity is <9.0 pmol Cy3 per g cRNA do not
proceed to the hybridization step.
Part 1
Step 1 One Colour Spike Mix:
Reagents: - 80
0
C in a black box
Agilent Spike Mix (thawed on ice)
Dilution Buffer (thawed on ice, but when used can be left at RT for 5 minutes)
- 1
st
dilution of Agilent Spike Mix serves as a positive control
- Can be stored for up to 2 months in -80
0
C and can be thawed up to 8 times. Make note on
the tube each time.
Starting
amount of
RNA
Serial Dilution Spike Mix
volume in
each rxn (l)
Total RNA
(ng) 1st 2nd 3rd
100
1:20 (2l Spike
Mix + 38l
Dilution Buffer)
1:25 (2l 1st
dilution + 48l
Dilution Buffer)
1:20 (2l 2nd
dilution +
38l Dilution
Buffer)
2
200
1:20 (2l Spike
Mix + 38l
Dilution Buffer)
1:25 (2l 1st
dilution + 48l
Dilution Buffer)
1:20 (2l 2nd
dilution +
38l Dilution
Buffer)
2

Use the sample to label chart to calculate the RNA concentration.
For 200 ng of RNA:
1
st
dilution: (Do if it needs to be made):
a. Label a 0.5 ml tube 1
st
dilution and date.
b. Flick thawed Spike mix to mix.
c. Heat at 37
0
C in circulating water bath for 5 minutes, wipe off tube to prevent
contamination, and flick once more.
d. Heat water bath to 40
0
C for cDNA step.
e. Spin briefly in microfuge.
f. Add 38l of dilution buffer to 1
st
dilution tube.
g. Add 2l of Spike mix to 1
st
dilution tube.
h. Vortex and microcentrifuge.
2
nd
dilution:
a. Vortex and quick-spin 1
st
dilution
b. Label a second 0.5 ml tube 2
nd
dilution
c. Add 48l of dilution buffer to the tube.
d. Add 2l of 1
st
dilution to the tube.
e. Vortex and microcentrifuge.
f. Use this only once and then discard.
3
rd
dilution:
a. Label a third 0.5 ml tube 3
rd
dilution.
b. Add 18l of dilution buffer to the tube.
c. Add 2l of 2
nd
dilution.
d. Vortex and microcentrifuge.
e. Use only once and then discard.

Step 2: Dilute total RNA sample:
Using the sample to label chart, determine how many ls it would take of your sample to get
200 ng of RNA. If the volume needed is less than 0.5l, then the sample is too concentrated and
requires a 1:3 dilution. Make sure that when you label the tube with the dilution with all the
information from its predecessor, along with the label 1:3 dilution and the date.
To dilute:
Add 3l of RNase-free water
Add 1l of the RNA sample
Spin briefly in mircocentrifuge.

Step 3: Preparing Labeling Reaction:
Reagents: -20
0
C in box labeled Agilent Labeling Reagents:
T7 promoter primer (GREEN cap; thaw on ice)
Nuclease-free water (WHITE cap; thaw at RT)
- Note that the total volume of RNA should not exceed 1.5l according to Agilent (p.23)
- It might be a good idea to set up a master mix, considering how small the volumes that
are aliquoted.
- Thaw the primer on ice
Step 3A: Promoter Ligation:
1. Start thermocycler to 65
0
C
2. Calculate how much water should be added to a 0.6 ml tube using the following formula:
2.5l total l RNA (No more than 1.5l)
2.5 is because we add 1l of water to the T7 primer + 1.5l of RNA
3. 200 ng of RNA should be aliquoted into the 0.6 ml tube. Mix by pipetting.
4. Add 2l of Spike Mix dilution to the tube. Total volume should be 4.5l
5. Add 0.8l of T7 promoter primer. Total volume should be 5.3l.
6. Incubate samples in thermocycler for 10 minutes. Jump to step 8 during this period.
7. Place samples on ice for 5 minutes

Step 3B: cDNA Synthesis:
Reagents: -20
0
C in box labeled Agilent Labeling Reagents:
- 5X 1
st
Strand Buffer (GREEN cap).
- 0.1 M DTT (WHITE cap; thaw on ice).
- 10mM dNTP (GREEN cap; thaw on ice. Contains RNA).
- AffinityScript RNase Block Mix (PURPLE cap. Thaw on ice. Contains enzymes).
(it might be a good idea to set up a master mix here).
8. Because there is so much salt in the 5X buffer, it crystallizes. To deal with this, prewarm
it at 80
0
C for 5-10 minutes in the old thermocycler on the bench. Leave it there until
needed. Be sure to flick and microcentrifuge when needed.
9. Spin samples in microcentrifuge.
10. Add 2l of 5X FS buffer to each sample. Because the buffer is really sticky, WIPE THE
SIDE of the tip before taking it out. (GREEN)
11. Add 1l of DTT to each sample. (WHITE)
12. Add 0.5l of dNTP to each sample (GREEN)
13. Add 1.2l of AffinityScript RNase Block Mix (PURPLE) to each sample. Very sticky,
so wipe the sides to avoid taking extra. Mix by pipetting. Total volume should be 10l.
14. Microcentrifuge briefly.
15. Incubate in water bath at 40
0
C for 2.5 hours. When done, dry tubes with Kimwipes
(Avoids contamination), and then quick-spin (makes sure samples are at the bottom of
tube).
16. Place in thermocycler at 70
0
C for 15 minutes (this deactivates the AffinityScript).
17. Place on ice for 5 minutes.
18. Microcentrifuge briefly.

STOPPING POINT HERE. STORE IN -80
0
C FREEZER.

Part 2
STEP 3C: cRNA Synthesis:
Reagents: -20
0
C in box labeled Agilent Labeling Reagents:
- Nuclease-free water (WHITE cap, thaw at rtp).
- 5X Transcription Buffer (BLUE cap. Thaw on ice).
- 0.1 M DTT (WHITE cap; thaw on ice).
- NTP mix (BLUE cap. Thaw on ice).
- T7 RNA polymerase Blend (RED cap. Thaw on ice).
- Cyanine 3-CTP (in foil bag. THAW ON ICE AND IN BAG).
(Master mix?)
1. Thaw samples and reagents.
2. Add each of the following to each sample and mix by pipetting:
a) 0.75l nuclease-free water
b) l 5X Transcription Buffer (BLUE)
c) 06l DTT (WHITE)
d) 1l NTP (BLUE)
Use Cy3 and T7 pipette for the last two:
e) 0.21l T7 RNA polymerase Blend (RED its an enzyme, so its really thick. Wipe tip
on sides before taking it out).
f) 0.25l Cy3 (FOIL BAG NOTE: This reagent is really sticky, so wipe sides of pipette
tip before taking out of tube. Also, its very photosensitive, so MOVE QUICKLY).
3. Quick Spin samples (Because they now have Cy3, they also need to be kept in the dark).
Total volume should now be 16l.
4. Place in water bath of 40
0
C for 2.5 hours. Cover lid and sides of water bath completely
with foil.
5. Wipe of sides with Kimwipes and quick spin.

STOPPING POINT HERE. STORE IN -80
0
C FREEZER.


Step 4: Purifying labeled/amplified RNA:
Set this up while samples are in step 3C.
1) Label 3 sets of 1.5 ml tubes per sample (two should be numbered in the order you set, the
other with all the information of the sample, the date, and the word label on it).
2) Add 84l of nuclease-free water to each sample and mix by pipetting. Transfer total
volume (100l) to first set of temporary 1.5 ml tubes.
3) Add 350l of RLT buffer and mix by pipetting.
4) Add 250l of absolute EtOH and mix by pipetting.
5) Get Qiagen Spin columns from 4
0
C fridge for each sample and label accordingly.
Transfer 700l of each sample to their respective spin columns. Note that this sample is
now really sticky so pipette up and down SLOWLY to get as much as possible. Throw
away old tube.
6) Centrifuge at 4
0
C for 30 sec at 13,000 rpm. Discard flow-through in Trizol waste. Filter
should look pink.
7) Add 500l of RPE buffer and spin again at 13,000 rpm at 4
0
C for 30 sec. Discard flow-
through.
8) Repeat previous step, but spin for 1 minute. Discard both flow-through and collection
tube.
9) Transfer column to second temporary tube. Cut off lid of column and centrifuge for 1
min. in 4
0
C at 13,000 rpm. Discard collection tube.
10) Transfer column to final, labeled tube and add 30l onto filter membrane. Seal column
with collection tubes lid, wait for 1 minute before spinning at 13,000 rpm at 4
0
C for 30
sec. discard column.
11) Keep cRNA on ice, covered with foil, and quantify.
Step 5: Quantification:
Click on microarray tab -> add 1.5l of nuclease-free water to start machine -> set to RNA->
Set dye 1 to Cy3 and dye 2 to none -> wipe off and add 1.5l of nuclease-free water and hit
blank -> wipe off and add 1.5l of samples, name it and hit measure.
Readings of importance: concentration of dye (pmol/l), 260/280, ng/l
Calculating yield and specific activity:
(Conc. of cRNA X 30l (elution volume) = g of cRNA
1000
(Conc. of Cy3/Conc. of cRNA) X 1000 = pmol of Cy3 per g of cRNA.