Microarray Labeling Protocol - 07-03-2012

8/11/2019 Microarray Labeling Protocol - 07-03-2012

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Edge labAgilent Microarray Labeling Protocol07/03/2012

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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


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Part 1

Step 1One Colour Spike Mix:

Reagents: - 800C 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)

- 1stdilution of Agilent Spike Mix serves as a positive control

- Can be stored for up to 2 months in -800C 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 + 38lDilution Buffer)

1:25 (2l 1st

dilution + 48lDilution Buffer)

1:20 (2l 2nd

dilution +

38l DilutionBuffer)

2

200

1:20 (2l Spike

Mix + 38lDilution Buffer)

1:25 (2l 1st

dilution + 48lDilution Buffer)

1:20 (2l 2nd

dilution +

38l Dilution

Buffer)

2

Use the sample to labelchart to calculate the RNA concentration.

For 200 ng of RNA:

1stdilution: (Do if it needs to be made):

a. Label a 0.5 ml tube 1stdilutionand date.

b. Flick thawed Spike mix to mix.

c. Heat at 370C in circulating water bath for 5 minutes, wipe off tube to prevent

contamination, and flick once more.d. Heat water bath to 40

0C for cDNA step.

e. Spin briefly in microfuge.

f. Add 38l of dilution buffer to 1stdilution tube.

g. Add 2l of Spike mix to 1stdilution tube.

h. Vortex and microcentrifuge.


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2nd

dilution:

a. Vortex and quick-spin 1stdilution

b. Label a second 0.5 ml tube 2nd

dilution

c. Add 48l of dilution buffer to the tube.

d.

Add 2l of 1stdilution to the tube.e. Vortex and microcentrifuge.

f. Use this only once and then discard.

3rd

dilution:

a. Label a third 0.5 ml tube 3rd

dilution.

b. Add 18l of dilution buffer to the tube.

c. Add 2l of 2nd

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.


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Step 3: Preparing Labeling Reaction:

Reagents: -200C in box labeled Agilent Labeling Reagents:

T7 promoter primer (GREENcap; thaw on ice)

Nuclease-free water (WHITEcap; 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 650C

2.

Calculate how much water should be added to a 0.6 ml tube using the following formula:2.5ltotal 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


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Step 3B: cDNA Synthesis:


- 5X 1stStrand Buffer (GREENcap).

-

0.1 M DTT (WHITE cap; thaw on ice).- 10mM dNTP (GREENcap; thaw on ice. Contains RNA).

- AffinityScript RNase Block Mix (PURPLEcap. 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 800C 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 10 l.

14.Microcentrifuge briefly.

15.Incubate in water bath at 400C 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 700C for 15 minutes (this deactivates the AffinityScript).17.Place on ice for 5 minutes.

18.Microcentrifuge briefly.

STOPPING POINT HERE. STORE IN -800C FREEZER.


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Part 2

STEP 3C: cRNA Synthesis:


- Nuclease-free water (WHITEcap, thaw at rtp).

- 5X Transcription Buffer (BLUEcap. Thaw on ice).

- 0.1 M DTT (WHITE cap; thaw on ice).

- NTP mix (BLUEcap. Thaw on ice).

- T7 RNA polymerase Blend (REDcap. 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 (REDitsan enzyme, so its really thick. Wipe tip

on sides before taking it out).

f) 0.25l Cy3 (FOIL BAGNOTE: 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 400C 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 -800C FREEZER.


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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 RLTbuffer and mix by pipetting.

4) Add 250l of absolute EtOH and mix by pipetting.

5) Get Qiagen Spin columns from 40C 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 SLOWLYto get as much as possible. Throw

away old tube.

6) Centrifuge at 40C for 30 sec at 13,000 rpm. Discard flow-through in Trizol waste. Filter

should look pink.

7) Add 500l of RPEbuffer and spin again at 13,000 rpm at 40C 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 40C 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 40C 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.

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Microarray Labeling Protocol - 07-03-2012