Ray Tabibiazar MD
Roger A. Wagner MD, PhD
Revised July 03
Labeling and Hybridization
to Stanford Mouse cDNA array
□
Place 30 ug
test RNA per reaction in each test tube. In separate tube, 30ug Reference RNA
per reaction.
□
Speedvac just to dryness. RNA may be
stored overnight at 80C at this point.
□
Resuspend RNA in 14.9 ul Primer mix containing 1 ul of 4 ug/ul
Anchor Primer (T20VN) and 13.9ul RNAse free
water. Pipet or vortex well to resuspend,
spin down for 15 seconds.
□
Incubate the reaction at 65-70°C at heat bloc for 10 min. (Prepare Labeling mix at this time.)
□
Transfer the tubes to ice.
Set up Labeling
Mix Vortex quickly, spin down.
* Add SSII to the mix last.
** Thaw the 5x
RT buffer at room temperature.
□
Add 15.1 uL of the reaction
mix to the annealed RNA on ice. Vortex to mix, spin 15 sec.
□
Incubate at 42 C for 60 min. Cover tubes with foil to
avoid bleaching dyes.
□
Add 1ul SSII (RT booster) to each sample. Vortex, spin.
□
Incubate at 42 C for additional 60 min.
□
Add 15 ul of 0.1N NaOH, 2mM EDTA
□
Incubate at 70 C for 30 min to degrade the RNA.
□
Neutralize by addition of 15 ul
of 0.1N HCl
Target
Purification using Qiaquick PCR purification kit
□
Add 500 ul
Buffer PB(Qiaquick) to each
labeling reaction and mix.
□
Combine the Cy3- labeled and
Cy5-labeled tubes into one.
□
Apply the sample (600 uL) to a Qiaquick PCR
purification column x 2 and centrifuge for 1 minute at full speed.
□
Discard the flow-through.
□
Wash with 700uL Buffer PE (EtOH added) to column and centrifuge 1 min at full speed.
Discard the flow-through.
□
Centrifuge 1 min at full speed (to
remove residue ethanol).
□
Place QIAquick
column in a clean 1.5-mL microfuge tube (not supplied
with the kit).
□
Add 40 uL
10 mM Tris-HCl, pH 8.5. to each tube.
□
Incubate at room temperature for 1
minute.
□
Centrifuge at full speed for 1
minute.
□
Add 40 uL
10 mM Tris-HCl, pH
8.5. to each
tube.
□
Incubate at room temperature for 1
minute.
□
Centrifuge at full speed for 1
minute.
□
Add 24ul polydA/tRNA/mouseCOT1
DNA mix to each purified probe.
□
Dry the sample just to dryness in
the Speedvac at medium heat (50°C). For 100 uL, it takes about 30 35 min.
Cover speedvac lid with foil to minimize light
exposure.
Prepare Hyb
□
Resuspend samples in 35 ul of Hyb Buffer (3.4X SSC, 0.3%SDS). Pipet up and down
thoroughly to fully resuspend
cDNA.
□
Incubate the samples at 98C for 2
minutes. (Bring water to boil, then remove from heat source. Begin incubation after boiling has stopped to
prevent tube caps from popping.)
□
Slow-cool the samples by spinning
14K RPM for 15 minutes at room temp
□
Add 35 ul to the mouse cDNA array and carefully place coverslip. (Add 15 ul 2X SSC buffer to
chamber wells to maintain humidity).
Place array in chamber and seal.
□
Incubate chambers in 65C water bath for 16-18 hrs
Wash arrays
□
Remove slide from chamber
carefully using plastic forceps. Hold
corner of slide near label with gloved fingers and gently swish the slide in
wash buffer #1 to remove coverslip
□
Put all slides in rack in wash buffer #1 (0.5X SSC, 0.01% SDS), and
then wash with gentle stirring for 5 min
□
Transfer slides to first wash
buffer #2 (0.06X SSC) and gently lift up and down 5X to remove SDS.
□
Transfer slides to 2nd
wash buffer #2. Wash with gentle stirring for 2 min.
□
Carry slides in wash #2 to
centrifuge
□
Quickly spin at 1,300 rpm for 2
minutes. (Sorvall)
□
Place slides in light proof
container, and scan as soon as possible.
Reagents:
Superscript II reverse transcriptase available from Biostores
100mM DTT included with SSII
5X RT buffer included with SSII
Unlabeled dNTPs available (RNAse free) from Biostores in
100mM concentration
Protocol to make stock mix:
100mM dATP 25ul
100mM dCTP 25ul
100mM dGTP 25ul
100Mm dTTP 10ul
H2O RNAse free 15ul
Total 100ul
RNAseOut ribonuclease
inhibitor Invitrogen Cat # 10777-019
Cy3 and Cy5 labeled dUTP
Amersham #PA55022 &PA53022 Order
3-4 each at a time. Stanford has
a significant ~30% discount, so ask for this when you order.
Mouse COT1 DNA 500ug (1ug/ul) Invitrogen
Cat # 18440-016 Available from Biostores. Store at
20C
PolydA (40-60) Amersham
Pharmacia Cat # 27-7988 5 A260
units. Order 4-5 at a time.
Resuspend PdA in 26 ul RNAse
free water for 10mg/ml stock. Store at 20
C.
Yeast tRNA available from Biostores
resuspend in RNAse free
water to 10mg/ml. Store
in aliquots at 20C.
NOTES:
We have
successfully used as little as 15ug total RNA for hybs
on the mouse microarray, but get optimal signal using
30ug, with little increased signal above that level.
Minimize
exposure to light by covering tubes with foil whenever possible to prevent Cy dye bleaching.
The GAPS II slides seem to be slightly heavier than regular
slides, perhaps due to the array label. It
is important to weigh the arrays in the carrier in which they will be spun
prior to hybridizing in order to prepare an appropriate balance carrier for
centrifugation.
Use 1.5 ml microcentrifuge tubes
for all steps. (We have had some trouble
with a newer brand of microcentrifuge tubes which are
more clear and transparent than the usual ones; these have proven brittle and break in
the Qiaquick steps.)
Use metal slide racks for centrifugation. (Shandon Lipshaw
#121) Glass racks are brittle and may break.
Stock polydA/tRNA/mouseCOT1 DNA mix
20 ul of Mouse Cot1 @1mg/ml, 2ul polydA @10mg/ml, & 2ul tRNA
@10mg/ml per array. Use 24ul of mix per array. Stock mixtures can be frozen down for later
use.
Stock
PolydA 5 A260units = 260ug, resuspend
each tube in 26ul to get 10mg/ml (PolydA (40-60) Amersham Pharmacia
Cat # 27-7988 5 A260 units. )
Wash Buffer #1
0.5X SSC
0.01% SDS
For 20
liters:
500 ml 20X
SSC
20ml 10% SDS
Bring to 20
liters with millipore H2O
Wash Buffer #2
0.06X SSC
For 20
liters:
60 ml 20X
SSC
Bring to 20
liters with Millipore H2O