Probe synthesis and Array hybridization:
Indirect Labeling:
cDNA Synthesis
1. Aliquot 20 g Total RNA per reaction to separate microtubes.
2. Add 5 l Oligo dT primer (0.5 g/l) to your RNA sample. *
3. Add DEPC H2O to 25 l total volume.
4. Heat tube at 70°C for 5 minutes
5. Prepare fresh dNTP mix if necessary
50X dNTP mix Recipe:
volume (ul)
[initial]
10 100 mM
10 100 mM
10 100 mM
4 100mM
6 100mM
dATP
dGTP
dCTP
[final]
25 mM
25 mM
25 mM
amino-allyl dUTP
dTTP
10 mM
15 mM
6. Prepare a Master Mix sufficient for the number of reactions plus one
extra reaction to ensure sufficient volume. Multiply quantities below
by the number of reactions:
Reagent
5X First Strand Buffer
10X dNTP mix
0.1 M DTT
DEPC ddH20
RNAsin (40U/ul)
Superscript II RT
(200U/ul)
7.
8.
9.
10.
11.
l per Reaction
10
5
5
2.5
0.5
2
Amount
Keep Master Mix at room temperature and continue.
Cool tube to 42°C, and then add 25 l of Master Mix.
Incubate at 42°C for 2 hour.
Add 17 l 0.5 M EDTA pH=8; 17 l 1N NaOH
Heat 70oC 10 minutes; Spin down brief
Add 43 l 1M Tris-HCl pH =7.5
Add 350 l sterile water (may store at -20oC overnight
Add entire sample to Micron Concentrator (Millipore Cat#42410)
Spin 12K rpm 7 min
Discard flowthrough
Add 400 l sterile water
Repeat two more times (3 spins Total; Cf an additional min on the
last spin)
19. Invert concentrator into a new tube to elute, Cf 2,000 rpm 2 min
20. Dry down sample speed vac (1 min per l on HIGH)
12.
13.
14.
15.
16.
17.
18.
Fluorescent Dye Coupling
Dye Preparation
* Cy3 or Cy5 reactive dye Packs (Amersham Biotech RPN5661)
Each pouch contains one tube of dye residue sufficient for 1
labeling reactions. Do not use if the desiccant packet is blue
1. Resuspend cDNA pellet in 15 l of nuclease free ddH2O
2. Resuspend Dye pellet in 15 l freshly prepared 0.1 M NaBicarbonate
Buffer pH 9.0
3. Add cDNA sample to dye soln. Mix well (do not vortex) and place the
tube at room temperature in the dark or wrapped in aluminum foil.
4. Incubate at room temperature for 1 hr
Quenching and Cleanup (Performed in the Hood)
1. Add 15 l hydroxylamine to each probe mixture
2. Let reaction incubate 15 min at room temp in dark
3. Add Cy3 Reaction to Cy5 Tube
QIAquick PCR Purification
*Use the QIAquick PCR Purification Kit Protocol to purify fragments of
100bp to 10kb from primers, nucleotides, polymerases, salts and
unincorporated dye.
*Add 100% EtOH to PE Buffer before use (see bottle label for vol)
*All Cf steps are >=10,000 x g in a table top microcentrifuge.
1. Add 5 vol (450 l) of PB Buffer to 1 vol (90 l) of combined labeled
probe.
2. Place a QIAquick spin column in the provided 2 ml collection tube
3. Place sample on column and cf for 30-60 sec.
4. Discard flow-through and replace column in same collection tube
5. Add 750 l of PE buffer, cf for 30-60 sec.
6. Discard flow-through and replace column in same collection tube
7. Cf an additional 60 sec.
8. Place filter into new tube
9. Add 50 l of ddH2O to the column, let stand for 1 min and cf at max 60
sec.
PPT Sample
1. Add 1l Poly-Acryl Carrier
2. add 5 l of Na Acetate (3M, pH5.2)
3. add 140 l of EtOH
4. Precipitate sample at –20oC 1 hr
5. Cf 20 min, 4oC13K rpm; Wash 100 l 70% EtOH; Air Dry ~10 min
Hybridization and Washing:
1. Warm Pre-hybridization buffer (5X SSC, 0.1% SDS, and 1% BSA) to
2.
3.
4.
5.
6.
7.
8.
Coverslip
size
22mmx60
mm
22mmx40
mm
42oC.
Warm 2X hybridization buffer (50% formamide, 10X SSC, and 0,2%
SDS) to 42 oC.
Place Slide into Coplin Jar containing pre-hybridization buffer (50 ml
42oC).
Incubate 40-45 minutes at 42oC.
Rinse slide by dipping 5X in ddH2O (Room Temp)
Dip slide in isopropanol; Spin 1 minute 1000 rpm in swinging bucket
rotor.
Use slide no later than 1 hr after pre-hybridization.
Check chart below for amount of sample for hybridization. Resuspend
Fluorescently labeled cDNA probe (from step 30) in sterile water. Add
Cot1 and oligo-dT. Do not add 2x hyb buffer yet. *
Array
Total
2X
Hyb vol Hyb
(ul)
Buffer
(ul)
2.2,3.1, 45
22.5
4.0
2.0,2.1 30
15
Sterile
water
volume
Cot1
Oligo
(1mg/ml) dT(0.5mg/ml)
15
2.5
5
7.5
2.5
5
9.
Denature probe 95oC 5 min, spin down quickly. Immediately add
appropriate amount of hyb buffer to mix.
10. Apply probe to slide. Cover with Glass Coverslip and incubate in
Corning Hybridization chamber overnight at 42 o Celsius. Heat
up 40 degree wash buffer 2 in preparation for tomorrow.
Wash and Scan:
1.
2.
3.
4.
5.
6.
Prepare 3 wash buffers
(a) Wash buffer 1 2X SSC; 0.1% SDS
Room Temp
(b) Wash buffer 2 0.2X SSC; 0.1% SDS
40oC
(c) Wash buffer 3 0.2X SSC
Room Temp
Remove coverslip by placing slide into low stringency Wash buffer 1
until the coverslip falls off.
Place into high stringency Wash buffer 2 and gently agitate for 5
minutes.
Place slide into Wash buffer 3 and gently agitate for 5 minutes.
Centrifuge Slide 3 minutes at 1000 rpm in swinging bucket rotor.
Scan
* - For amplification, Steps 3 and 37 are changed. The RT (step 3) is primed with
Random Hexamers, equal amount (in ng) as starting total RNA before
amplification. The blocking (step 37) you need to add 2 g of T7-oligo-dT as
blocker, decrease water accordingly.