MODULE 3 – Day 5
Label the mRNA isolated from HeLa cells transfected
with siRNAs targeted to the EGFP, p53, ATM, ATR,
EXO1 and AAG gene transcripts and hybridize to DNA
microarray slides
MOST STUDENTS WILL WORK IN GROUPS OF THREE
The total RNA from your previous transfection experiments has been isolated for you over the
weekend. For the two class sections combined, 8 chips total will be hybridized in order to do compare
the gene expression profiles of the following 8 sets of cells:
1.
2.
3.
4.
5.
6.
7.
8.
EGFP knockdown vs. oligofectamine-only treated cells
p53 knockdown vs. oligofectamine-only treated cells
ATM knockdown vs. oligofectamine-only treated cells
ATR knockdown vs. oligofectamine-only treated cells
Exo1 knockdown vs. oligofectamine-only treated cells
AAG knockdown vs. oligofectamine-only treated cells
oligofectamine-only vs. oligofectamine-only treated cells
untreated vs. oligofectamine-only treated cells
Chip # 7 (above) will control for cDNA synthesis and microarray hybridization effects. Chip # 8 will
control for effects specific to oligofectamine treatment. Each group is responsible for completing one
of the 8 microarray experiments, and the data will be shared with the entire class. Each group will
reverse transcribe mRNA from each of their two cell populations, label the cDNA, and hybridize the
cDNA to a DNA microarray (one microarray per group).
Before you begin, take note of the following:

RNase contamination is everywhere! You must use gloves, and spray your benches with
RNase away. Wipe off pipettes with RNase away as well.

The dyes that are used are light sensitive, and must be shielded from light. Protect them from
degradation by wrapping tubes in foil.

Do not touch the surface of the DNA microarray or the DNA will be wiped off.
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
You will be working with very small volumes.
o When pipetting a reagent into a tube, make sure you bring the pipette tip all the way to
the surface of the liquid and depress the pipette fully.
o Mix ONLY by tapping the eppendorf tube gently with your finger.
o Make sure all of the liquid is at the bottom of the tube. If it is not, spin the tube in the
microfuge by holding down the start/stop button for several seconds and then pressing
it again to stop it.
o Spin also after high temperature incubation steps because some of the liquid may
evaporate from the bottom and condense at the top of the tube. (*quick spin* is noted in
the procedure everywhere it’s important)
Required Materials:
For the cDNA Synthesis Part I (all on ice):
25 g total RNA from knockdown or control cells
25 g total RNA from oligofectamine treated
cells.
{Oligod(T)18 + alien mRNA spikes}
Cy3 dye
Cy5 dye
Master Mix
Super Script II Reverse Transcriptase (200U/ml)
For the cDNA Synthesis Part II (at room temp):
{0.5M EDTA + 1N NaOH*}
1M Tris, pH 7.5
For the Prehybridization:
1 Microarray chip (per group)
1 Lifter slip
1 Hybridization chamber
Prehybridization buffer
Filtered dH2O in 50 ml conical tube
100% Isopropanol in 50 ml conical tube
For the cDNA Clean-up (at room temperature):
cDNA from cDNA synthesis step
1 Qiagen column (per group)
Buffer PB
Buffer PE
dH2O pH7
1 eppendorf tube
*equivalent to 1M NaOH
For the Hybridization:
cDNA from cDNA clean-up step
Hybridization buffer
1 Hybridization chamber
1 lifter slip
ddH2O
1 Ziploc bag
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cDNA Synthesis and Hybridization Procedure (read each task
entirely before starting).

Preparation
1. Establish “RNA ONLY” benchtop, eppendorf tubes and pipettes.
2. Wipe gloves, pipettes and surfaces down with RNase away before proceeding.

cDNA Synthesis Part I.
You will begin with your 2 eppendorfs, each containing 25 g of total RNA, one from the siRNA
knockdown, one from the control cells. Add reagents to these two eppendorfs according to the
following steps.
1. Add 2 l {Oligod(T)18 + alien mNRA spikes} to each of your 2 samples.
2. Incubate at 65°C for 10 min. (*quick spin*)
3. Incubate at room temp. (25°C) for 5 min.
Prepare for light-sensitive Cy dyes by putting foil over your tube rack. From this point on, protect
your samples from light exposure at all times.
4. Add 4 l Cy3 dye to oligofectamine-treated control sample.
5. Add 4 l Cy5 dye to knockdown sample (oligofectamine-treated/untreated for 7 & 8).
6. Incubate at 42°C for 2 min.
7. Add 8.6 l Master Mix to each of your 2 samples.
8. Add 2 l Super Script II RT to each of your 2 samples. **This step is VERY IMPORTANT –
make sure that you see the 2 l get pipetted directly into the liquid.** (*gentle tapping +
quick spin*)
9. Incubate at 42°C for 60 min. (*quick spin*)

Prehybridization of Chip.
Remember, do not touch the surface of the DNA microarray or the DNA will be wiped off. ONLY
hold the glass slide by its edges or near the ends, away from the array in the middle. The glass
slide has been etched on the side that has the microarray.
1. Place the chip **array-side up** in the bottom of the hybridization chamber
2. Place lifter slip over the array **with the space between the array and the slip**.
3. Add 30 l prehybridization solution to edge of lifter slip on top of array. The solution should
get sucked under the slip and cover the entire array area through capillary action.
4. Close and seal the hybridization chamber.
5. Incubate at room temp for 45 minutes.
6. Wash slide by placing slide in filtered dH2O in conical tube. Shake gently until lifter slip
comes off. Wash for 2 minutes. **Start timing AFTER the lifter slip comes off.**
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7. Wash slide in 100% isopropanol in conical tube for 2 minutes.
8. Air dry slide (wick with kimwipe – do NOT touch the array with the kimwipe!!).

cDNA Synthesis Part II.
Your 2 eppendorfs, containing mRNA that you reverse transcribed and labeled, should be done
incubating (step 9 from cDNA synthesis Part I). Don’t forget to protect them from light!
1.
2.
3.
4.
5.

Cool on ice for 5 min.
Add 6 l {0.5M EDTA + 1N NaOH} to each of your 2 samples.
Incubate at 70°C for 15 min. (*quick spin*)
Cool on ice for 5 min.
Add 7.5 l Tris pH 7.5 to each of your 2 samples.
cDNA Clean-up.
You will now purify the cDNA that you synthesized and labeled in the previous steps. Each group
will use one QIAgen column.
1. Add 250 l Buffer PB to each of your 2 samples. (*quick spin*)
2. One at a time, load both of your 2 samples onto the QIAgen column (285 l each). **Try as
much as possible to load equal amounts of both samples. You should have 293.5 l of each if
everything was perfect.
3. Spin at maximum speed in microfuge for 1 minute.
4. Discard eluant (liquid that flowed through, which is in the collecting tube that the QIAgen
column sits in).
5. Add 650 l Buffer PE to the QIAgen column
6. Spin at maximum speed for 1 minute.
7. Discard eluant.
8. Spin at maximum speed for 1 minute to get rid of all PE.
9. Discard eluant.
10. Transfer the QIAgen column to a new, labeled eppendorf tube.
11. Elute the cDNA by adding 30 l dH2O pH7 directly onto column membrane. **It is crucial
that the water is added to the membrane – the white circle in the middle of the column bottom
– NOT to the sides of the column, and NOT to the exposed edge around the membrane.
12. Incubate for 2 minutes at room temp.
13. Spin at maximum speed for 1 minute.
14. Discard the column, keeping the eppendorf tube containing your eluted cDNA.
15. Add 30 l of hybridization buffer to the tube containing your cDNA.
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
Hybridization.
You will now hybridize the cDNA that you synthesized, labeled and purified in the previous steps,
to a DNA microarray. Each group will use one microarray.
1.
2.
3.
4.
5.
6.
Place the chip **array-side up** in the bottom of the hybridization chamber
Place lifter slip over the array **with the space between the array and the slip**.
Denature your cDNA by placing tube in 80C heat block for 2min.
Chill on ice for 2min.
Spin at maximum speed for 10 seconds.
Slowly apply your cDNA in hybridization solution (50 ul) to edge of slip. Capillary action will
draw solution over the array.
7. Add 10l ddH2O to both reservoirs in chamber.
8. Cover and reassemble hybridization chambers. **very important that it is sealed properly**
9. Place chambers in Ziploc bag(s)
10. Incubate in 42C light proof water bath. Weigh down bag(s) with a plastic tube rack.
After 16 hours of hybridization, the microarrays will be washed for you. You will scan them
during the next class period.
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