Liu Lab protocol
Updated on November 1, 2001
by Amir Jazaeri
Jazaeria@mail.nih.gov
Preparation of Probe and
Microarray Hybridization
Using Amplified RNA
This
protocol is designed for prints with a 40 x 22 mm2 spot
area
(1) |
Random
primers |
|
Invitrogen |
Cat# 48190-011 |
(2) |
Cy3-dUTP |
1 ml |
NEN
Life Science |
Cat# NEL999 |
(3) |
Cy5-dUTP |
1 ml |
NEN
Life Science |
Cat# NEL999 |
(4) |
SuperScript II |
200 U/µl |
GIBCO-BRL |
Cat# 18064-014 |
(5) |
RNAsin |
20-40 U/µl |
Promega |
Cat# N2515 |
(6) |
Yeast total tRNA |
4 µg/µl* |
Sigma |
Cat# R8759 |
(7) |
human
COT-1 DNA |
500µl (1µg/µl) |
Roche |
Cat# 1-581-074 |
(8) |
polyA |
8 µg/µl* |
Sigma |
Cat# P-9403 |
(9) |
100
mM dNTP set |
20X, low-dT^ |
Amersham-Pharmacia |
Cat# 27-2035-01 |
(10) |
Microcon |
YM-30 columns |
Amicon |
Cat# 42410 |
(11) |
BSA |
|
Sigma |
Cat#B4287 |
Other reagents: 20X SSC, TE pH7.4, 10% SDS, 500 mM
EDTA, 1M NaOH,
1M Tris-HCl pH7.5, sterile dH2O
* comes lyophilized,
must be resuspended at specified concentration.
^ comes
as 100 mM;
for 20X stock 10 mM each of dATP, dGTP, dCTP and 4 mM of dTTP.
For a total volume of 250 ml:
25 ml dATP + 25 ml dGTP + 25 ml dCTP + 10 ml dTTP + 165 ml H2O
I. Probe Preparation
1.
Prepare the following RT labeling reaction mix for each probe:
Component |
Vol (µl) |
Random primer (3 µg/µl) |
2 |
5-6 µg amplified RNA + H2O |
17 |
Incubate at room
temperature for 10 min.
2. Add
the following mix to each probe:
Component |
Vol. (µl) |
5X first strand buffer |
8 |
20X lowT-dNTP mix |
2 |
0.1 M DTT |
4 |
RNAsin |
1 |
Cy-3 or Cy-5 dUTP |
4 |
SuperScriptII enzyme |
2 |
Incubate at 42°C for 60 min.
3. Add 5 µl of 500 mM EDTA. STOP
REACTION WITH EDTA BEFORE ADDING NaOH!!
4. Add 10 µl of 1M
NaOH.
5. Incubate at 65oC
for 15 min. to hydrolyze residual RNA.
6. Cool to room
temperature.
7. Add 25 µl of 1 M
Tris-HCl (pH7.5) to neutralize pH.
1.
Add 500 µl of 1XTE to Microcon-YM30 column and spin at 13Krpm
for 5-6 min. to wash the column. Check for membrane integrity by looking into
the top insert. A thin film of TE (~50 ml)
should just cover the membrane.
2. Add 400µl 1XTE to each of the sample tube (from step 7 in I) and transfer all contents to the washed Microcon-YM30 column.
3. Spin at 13Krpm for 5-6 min. until ~50 µl is left on the membrane. Check for the “crystals of Sotiriou” (dye crystals along the edge of the column membrane), indicating the probe is likely to be good.
4. Add 450µl 1XTE to column; spin down to ~50µl as above. Again, check for the crystals. Invert the Cy-3 labeled probe into a clean tube, and spin at 14K for 1 min. to elute the probe.
5. Carefully add the Cy-3 labeled probe to the Cy-5 labeled probe in the column. Add ~450 µl 1XTE to column and spin at 13Krpm until ~13-14 ml of combined probe remains on the membrane (check with pipette).
6. Invert the combined probe into a clean tube, and spin at 14K for 1 min. to elute.
7. Transfer the probe (14 ml) into a clean eppendorf tube and store at 4 oC until ready to hybridize.
III. Probe Hybridization
1. Prepare Prehybridization Buffer (5X SSC, 0.1% SDS, 1% BSA), store in aliquots at ‑20 oC and warm to 42oC before use:
Component |
Vol. (µl) for 1ml |
20X SSC |
250 |
20% SDS |
5 |
100mg/ml BSA |
100 |
H2O |
645 |
Add 20 ml of H2O
to each humidifying well in the Hybridization Chamber (to maintain humidity). Place
40 ml of prehybridization buffer to the
center of the slide and quickly (but carefully!) place the cover slip on the
slide, taking care to prevent bubble accumulation beneath slip. Firmly attach
margin clamps of the Hybridization Chamber, and incubate at 42 oC for least 1 h.
2. Wash slide in distilled H2O for 2 min. followed by isopropanol for 2 min. Dry slide in centrifuge (5804R, Eppendorf) at 705 rpm (~70x g) for 4 min. Proceed with step 4 and set up probe for hybridization. It is advisable to hybridize the slide within 1 h of the prehybridization step.
3. Make fresh 2X Hybridization Buffer (50% formamide, 10X SSC, 0.2% SDS):
Component |
Vol. (µl) |
20X SSC |
50 |
formamide |
50 |
10% SDS |
2 |
Keep 2X Hybridization Buffer
at 42 oC.
4. Mix probe (from II, step 7) with 2 µl COT1-DNA (1 µg/ml), 2 µl polyA (8-10 µg/ml), and 2 µl yeast tRNA (4 µg/ml). Denature for 1 min. at 100oC, and snap cool on ice and spin down.
5. Add 20 µl of 2X hybridization buffer to the denatured probe, mix well (take care to minimize bubble formation) and keep at 42 oC until ready to spot on slide.
6. Prepare hyb chamber as in the prehybridization step with 20ml of distilled water in each well. Place slides in chambers face up.
7. Hybridize slide with probe 14-16hrs at 42 oC.
IV. Slide Washes
1.
Carefully
remove margin clamps of Hybridization Chamber to prevent water from seeping in
and contaminating the array.
2.
Remove
slide from chamber, hold slide with forceps and allow cover slip to fall off
into the solution comprising 2X SSC, 0.1% SDS.
3.
Wash
for 4 min. in 1X SSC, 0.1% SDS.
4.
Wash
for 4 min. in 0.2X SSC.
5.
Wash
for 1 min. in 0.05X SSC.
6.
Quickly
spin dry in centrifuge at 705 rpm (~70x g)
for 4 min. If water droplets can be
seen on the slide spin for another 4 minutes.
7.
Minimize
exposure to light by placing the dried slides in a slide box until ready for
scanning.
Washes: |
2X SSC, 0.1%SDS |
1X SSC, 0.1%SDS |
0.2X SSC |
0.05X SSC |
20X SSC: |
200 ml |
100 ml |
2 ml |
0.5 ml |
20%SDS: |
10 ml |
10 ml |
- |
- |
dH2O |
|
Make up to 2 L |
|
|