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Edge Lab Agilent Microarray Labeling Protocol 6/18/12

~6.5-7 hours (including 1 hour to allow samples to thaw)


Notes before Beginning:
The water bath and thermalcyclers take ~5 minutes to come to temperature.
Allow samples to thaw on ice from -80C for 1 hour.
Thaw labeling reagents from -20C on ice before use. When reagents have thawed to liquid,
mix briefly on vortex, then quick spin for 5 s. Reagents that must be kept on ice at all times.
Agilent Labeling kit requires input RNA range between 10-200 ng of total RNA.
All reagent/enzyme product numbers listed at end of protocol.
Cy3 is photoreactive; when working with it, work quickly and keep dye and labeled samples
covered with aluminum foil. There are small cracks allowing light through the lid of the
circulating water bath so place a sheet of foil over the lid when incubating labeled samples.
Be sure to wipe tubes off when removing them from the water bath or ice to prevent
contamination.
Always look at the reagent volumes when taking tubes out of the freezer to make sure you have
enough and will not run out in the process.
After using the reagents make a note of how many samples you used them in.


Pipetting:
Since we are working with such small reaction volumes, every droplet matters. The success of
the labeling reaction is dependent on the exact concentrations of reagents present.
Remember that there are two stops on the pipette. Press down to the first stop, put pipette tip
in the liquid, then release to suck up. Press down to the second stop to dispense into your tube.
Hold your tubes up close to visually confirm the reagent is being sucked up, then dispensed.
Mix up and down thoroughly by pipetting to ensure complete mixture of all reagents.
Make sure the tip is securely attached to the pipette.
Make sure the lid of the tube is open all the way so it doesnt touch the shaft of the pipette. If
this happens, wipe it off with RNase Away before continuing.
If, after pipetting, a bubble remains in the pipette tip, pipette gently up and down until the
bubble is gone. If this isnt successful then try removing the tip, putting it back again and
pipetting down the air within the tip.
When pipetting a sequence of reagents, keep track if the reagents and the sample tubes by
changing their location to avoid confusions, e.g. from the ice to a tube race or from one corner
of your ice bucket to the other. Move each tube after adding each reagent.
After using each reagent tube, immediately close the lid firmly and put back on ice.
Pipette slowly and make sure there arent any remaining droplets in the tip before discarding it.
When sucking up the reagent from the stock tube, wipe pipette tip off on the side of the stock
tube. Reagents sometimes stick to the outside of the tip and you do not want to add any more
than the intended volume.




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Step 1: Prepare One Color Spike Mix (~0.5 hours)

Reagents, stored at -80C
Agilent Spike Mix
Dilution Buffer

-Store Agilent RNA Spike-In Kit, one color in -80C for up to 1 year
-1
st
Dilution of Agilent Spike Mix positive controls can be stored up to 2 months in -80C and can be
frozen/thawed up to 8 times. So, this step is not necessary every time.

Table 1 (For full table calculations, see table 1, pg 19 of Agilents protocol)
Starting
Amount
Of
RNA
Serial Dilution Spike Mix
Volume in
Each rxn
Total RNA
(ng)
1
st
2nd 3rd
100 1:20
(2L Spike Mix+
38L Dilution
Buffer)
1:25
(2L 1
st
dilution +
48L Dilution
Buffer)
1:20
2L 2
nd
dilution +
38L Dilution
Buffer)
2
200 1:20
(2L Spike Mix+
38L Dilution
Buffer)
1:25
(2L 1
st
dilution +
48L Dilution
Buffer)
1:10
2L 2
nd
dilution+
18L Dilution
Buffer)
2

Be sure to calculate starting RNA concentration accordingly.

For 100 ng of total RNA starting sample:
1) Create the 1
st
dilution:
a. Label a 1.5 mL tube Spike Mix 1
st
dilution
b. Vortex thawed Spike Mix
c. Heat at 37C in circulating water bath for 5 minutes & vortex Spike mix again (note: be sure
to wipe tubes off after removing them from water bath to prevent contamination)
d. Heat water bath up to 40C
e. Spin briefly in microcentrifuge
f. Add 2 L of Spike Mix to 1
st
Dilution tube
g. Add 38 L of Dilution Buffer provided in Spike-In kit (1:20)
h. Vortex & microcentrifuge

2) Create 2
nd
dilution
a. Label new 0.5 mL tube Spike Mix 2
nd
dilution
b. Into 2
nd
dilution tube, add 2 L of 1
st
dilution
c. Add 48 L of Dilution Buffer (1:25)
d. Vortex & microcentrifuge
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Use once and discard.

3) Create 3
rd
dilution
a. Label new 0.5 mL tube Spike Mix 3
rd
dilution
b. Into 3
rd
dilution tube, add 2 L of 2
nd
dilution
c. Add 38 L of Dilution Buffer (1:20)
d. Vortex & microcentrifuge

Use once and discard.

For 200 ng of total RNA starting sample:
1) 1
st
dilution:
a. Label a 1.5 mL tube Spike Mix 1
st
dilution
b. Vortex thawed Spike Mix
c. Heat at 37C in circulating water bath for 5 minutes & vortex Spike mix again (note: be sure
to wipe tubes off after removing them from water bath to prevent contamination)
d. Heat water bath up to 40C (for step 14).
e. Spin briefly in microcentrifuge
f. Add 2 L of Spike Mix to 1
st
Dilution tube
g. Add 38 L of Dilution Buffer provided in Spike-In kit (1:20)
h. Vortex & microcentrifuge


2) 2
nd
dilution:
a. Label new 0.5 mL tube Spike Mix 2
nd
dilution
b. Into 2
nd
dilution tube, add 2 L of 1
st
dilution
c. Add 48 L of Dilution Buffer (1:25)
d. Vortex & microcentrifuge

Use once and discard.


3) Create 3
rd
dilution
a. Label new 0.5 mL tube Spike Mix 3
rd
dilution
b. Into 3
rd
dilution tube, add 2 L of 2
nd
dilution
c. Add 18 L of Dilution Buffer (1:20)
d. Vortex & microcentrifuge

Use once and discard.

Step 2: Dilute total RNA sample (~15 min)
If RNA samples are too concentrated to add 200 ng in a reasonable volume (<0.5 L), make a 1:3
dilution in a new 0.6 mL tube. Label that tube with the same original information, plus the date and
diluted and save in case you need it again in the future.

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Add 3 L RNase-free water.
Add 1 L RNA sample.
Spin samples briefly in microfuge.

Step 3: Prepare Labeling Reaction (~5.5 hrs)

Reagents stored in -20C.
- T7 promoter primer (green cap)
-Nuclease-free water (white cap)

3A: Promoter Ligation (20-30 minutes)

1) After appropriate dilutions have been made, add the necessary volume of cold RNAse-free water to
a new 0.6 mL tube. This amount of water is calculated based on the volume of RNA added
according to this calculation = 2.5 L - L RNA volume (2.5L comes from the Agilent protocol
instructions to add 1L of water to the T7 promoter primer + 1.5L RNA p. 24)
2) Add the necessary volume of RNA according to calculations to obtain 200 ng of sample. According
to Agilents protocol, the volume of RNA should not be more than 1.5 L (p. 23). Mix by pipetting.
3) Add 2 L of 3rd diluted Spike Mix to each tube. Each tube now contains volume of 4.5 L.
4) Add 0.8 L T7 promoter primer to each sample. Each tube now contains a total volume of 5.3 L.
5) Denature primer & template by incubating the reaction at 65C in thermocycler for 10 minutes. In
the meantime you can jump to step 7 and then come back to step 6 when the 10 minutes are over.
6) Place reactions on ice for 5 minutes.

3B: cDNA Synthesis (~2.5 hours)

Reagents stored in -20C.
-5x 1
st
strand buffer (green cap) (also aliquoted into 0.6 mL tube)
-0.1 M DTT (white cap)
-10 mM dNTP (green cap)
- AffinityScript RNase Block Mix (violet cap)

7) The 5X first strand buffer is a salt that tends to crystalize. In order to use it you need to prewarm it
at 80C for 5 to 10 minutes (use the old thermocycler on the central bench, the tube doesnt fit, but
you can lay it down). Vortex & microcentrifuge. Keep at room temp until needed.
8) Spin samples in microcentrifuge briefly.
9) Add 2 L of 5x 1
st
strand buffer to each sample. The buffer is sticky so wipe the side of the pipette
tip in the tube before taking it out. Remember to keep track of the reagents location.
10) Add 1 L of 0.1 M DTT to each sample.
11) Add 0.5 L of 10 mM dNTP to each sample.
12) Add 1.2 L of Affinity Script RNase Block mix (purple cap) to each sample. This reagent is very
sticky and limiting. Be very careful of not to take extra. Mix by gently pipetting. Each sample now
has total volume of 10 L.
13) Microcentrifuge samples briefly.
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14) Incubate samples at 40C in circulating water bath for 2.5 hours. Dry tubes with Kimwipe and Quick
spin them to make sure there is no evaporative loss and the entire sample is at the bottom of the
tube.
15) Place samples in 70C thermocycler for 15 minutes (deactivates Affinity Script enzyme). Place on
ice for 5 minutes.
16) Spin samples in microcentrifuge.

<STOP> if you do not immediately continue to next step, store samples I the -80C freezer.

2C: cRNA synthesis (at least 2 hours)

Reagents stored in -20C.
-Nuclease-free water (white cap)
-5X Transcription Buffer (blue cap)
-0.1 M DTT (white cap)
-NTP mix (blue cap)
-T7 RNA Polymerase Blend (red cap)
-Cyanine 3-CTP

17) To each sample add and mix by gently pipetting.
a. 0.75 L Nuclease-free water
b. 3.2 L 5X Transcription Buffer
c. 0.6 L 0.1M DTT
d. 1 L NTP mix

And for the last 2 reagents only use the special pipette labeled Cy3 and T7 only.

e. 0.21L T7 RNA Polymerase Blend (This reagent is very thick so wipe the pipette tip down
before taking it out of the tube.)
f. 0.25 L Cyanine 3-CTP (This reagent is super sticky. Keep it in the bag until needed and spin
before using it. After adding this reagent, the sample has to be kept in the dark as well.)

18) Quick spin the samples. Each one now contains a total volume of 16 L.
19) Cover water bath with foil and incubate samples in circulating water bath at 40C for 2.5 hours.
Turn the water bath off.

<STOP> if you do not immediately continue to next step, store samples in -80C

Step 3: Purify labeled/amplified RNA (0.5 hrs) similar to Qiagen Purification method

20) Label two sets of 1.5 mL tubes per sample. One set is temporary and the other isnt such that you
should label all the samples information on the lid and on the side of the tube.
21) Add 84 L of nuclease-free water to cRNA sample. Mix by pipetting gently and transfer the entire
volume to a new temporary 1.5 mL tube. Now, the total volume equals 100 L.
22) Add 350 L of Qiagen Buffer RLT & mix by pipetting. (Note there are 2 buffers! For this step use RLT
only.)
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23) Add 250 L of 100% EtOH & mix by pipetting. Do NOT centrifuge.
24) Label with the temporary name a Qiagen spin column per sample. The columns are stored in the -
4C fridge. Then, transfer 750 L the mix from previous step to a Qiagen spin column in 2 mL tube.
The sample will be sticky so pipette up and down very slowly in order to take the most of the
sample you can. Do your best, but keep in mind that little droplets in the walls of the old tube are
expected. Discard old tube.
25) Centrifuge sample at 4C for 30 s at 13,000 rpm. You can leave the centrifuges inner lid out
between steps because you will use it right away. However, close the outer lid to keep the
temperature at 4C. Discard flow-through into Trizol waste bin. The filter should look pink.
26) Add 500 L of buffer RPE (diluted with EtOH (if it isnt diluted yet, dilute it first) to the column.
Centrifuge the sample at 4C for 30 s at 13,000 rpm (*or at room temperature is ok too). Discard
flow-through, but re-use the collection tube.
27) Add additional 500 L of buffer RPE (the diluted one again) to column. Centrifuge sample at 4C for
60 s at 13,000 rpm *. Discard flow-through & collection tube.
28) Transfer RNeasy column to new and temporarily labeled 1.5 mL tube, cut the lid off & centrifuge it
at 4C for 60 s at 13,000 rpm. Discard this collection tube & use a fresh tube to elute the clean
cRNA sample.
29) Elution: transfer RNeasy column to a new 1.5 mL collection tube labeled with all the samples info.
Add 30 L RNase-free water onto filter membrane, close the lid and wait 60-90 s. Then, centrifuge
at 4C for 30 s at 13,000 rpm*. Discard the column. The sample is at the bottom of the 1.5 mL tube.
30) Keep cRNA sample on ice and covered from light until quantification.

Step 4: Quantification

31) Use the ND 1000 software (Nanodrop) and the spectrophotometer. Hit Microarray RNA
401Cy3 add 1.5 L of RNase free water hit Ok wipe down the water add another 1.5
L of RNase free water hit BLANK wipe down the water add 1.5 L of sample, make sure
you name it hit MEASURE wipe down the sample and add the next one. Save the information
on an Excel sheet in your folder and print it.

-While quantifying, record dye concentration (pmol/ L); 260/280, ng/L

-To determine yield & specific activity of each reaction:

(Concentration of cRNA x 30 L (elution volume) = g of cRNA
1000


Concentration of Cy3 x 1000 = pmol Cy3 per g cRNA
Concentration of cRNA






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Yields & Specific Activity
Microarray Format Yield (g) Specific activity
(pmol Cy3 per g cRNA)
1-pack

5 6
2-pack 3.75

6
4-pack 1.65 6
8-pack 0.825 6


Agilent Product Numbers for Reagents & Enzymes

Low Input Quick Amp Labeling Kit, One Color Agilent p/n 5190-2305
T7 primer, 24 L
5X 1
st
Strand Buffer, 100 L
0.1M DTT, 70 L
10 mM dNTP Mix, 20 L
AffinityScript RNase Block Mix, 36 L
5X Transcription Buffer, 160 L
NTP Mix, 35 L
T7 RNA Polymerase Blend, 10 L
Nuclease-free water, 250 L
Cyanine 3-CTP

RNA Spike-In Kit, One Color (3 vials) Agilent p/n 5188-5282
Spike Mix, 10 L
Dilution Buffer, 1.2 mL

RNeasy Mini Kits (50 columns) Qiagen p/n 74104
50 spin columns
Buffer RPE
Buffer RLT