Probe preparation and hybridization
Probe preparation
Prepare in two 200 µl PCR tubes:
2 µg oligo-dT (21 mer)
2 µg mRNA (CTL or TR)
in 13.4 µl H2O
Heat 5 min, 70°C (PCR machine).
Leave 5 min at RT.
To each tube add:
6 µl 5X SuperScript II buffer
3 µl 0.1 M DTT
3 µl Cy3-dCTP (1 mM) or Cy5-dCTP (1 mM) (Amersham Cat. # PA53021, PA55021)
0.6 µl dNTPs (25 mM dATP, dTTP, dGTP; 10 mM dCTP)
2 µl SuperScript II Reverse Transcriptase (Gibco BRL Cat. # 18064-14)
2 µl RNAse Inhibitor (15 U/µl) (Gibco BRL Cat. # 15518-012)
Incubate 1 hr, 42°C (PCR machine).
Pool the two tubes (CTL + TR)
Add:
2.65 µl 25 mM EDTA
3.3 µl 1 M NaOH
Incubate 10 min, 65°C (PCR machine).
Add:
3.3 µl of 1 M HCl
5 µl of 1 M Tris pH 6.8
Purify probe with Qiagen MinElute PCR purification kit (# 28004)1
Mix 5 volumes of buffer PB with 1 volume of probe, apply to MinElute column
Spin1 1 min (>10'000 x g). Discard flow-through.
To wash, add 750 ml buffer PE to the MinElute column. Centrifuge for 1 min
Discard flow-through and centrifuge the column for an additional 1 min.
To elute DNA, place the MinElute column in a clean microcentrifuge tube. Add 10 ml buffer EB (10 mM Tris.Cl, pH 8.5) to the center of the membrane, let the column stand for 1 min, and then centrifuge for 1 min. Average eluate volume is 10 ml2.
Measure dye incorporation with 1 ml of probe with a NanoDrop instrument
Mix in a a 200 µl PCR tube (for a 22x22mm coverslip):
9 µl labeled probe
1.9 µl 20X SSC (3X final)
1.25 µl Yeast tRNA 2 µg/µl (0.2 µg/µl final)
0.5 µl 10% SDS (0.4% final)
Adjust volumes dpending on the size of the coverslip
Proceed to hybridization
Hybridization.
Put cDNA array slide in hybridization chamber (TeleChem International Cat. # AHC-1*).
Add 10 µl of 3X SSC in the two grooves at both ends of the slide to humidify chamber:
Heat probe 1 min, 95°C.
Spin 1 min, max speed.
Rapidly add probe to slide3, lay cover slip slowly on top of solution4.
Close chamber, immerge into 64°C bath. WORK QUICKLY.
Hybridize from 6 hrs to O/N without agitation.
Washes
Dismount chamber, put slide in glass slide holder with coverslip5.
Place slide holder in glass dish containing washing solutions (ca 500 ml), move slide holder up and down during washing time for good agitation then transfer to next dish. Alternatively use Microarray Wash Station (TeleChem International Cat. # AW-1*).
-Washes (RT):
5 min 2X SSC, 0.1% SDS
5 min 2X SSC, 0.1% SDS
1 min 0.2X SSC
1 min 0.2X SSC
1 min 0.1X SSC
1 min 0.1X SSC
-Dry slide in centrifuge 2 min, 2650 rpm.
-Store in light-tight box until scanning6.
Notes:
1. All washing and centrifugation steps are done at room temperature. Chilling results in precipitation of free label. Avoid putting the probe on ice at any time.
2. The eluate should be slightly purple. This is an indication of good labeling.
3. Apply probe where genes are located (use provided mask).
4. Cover slips must be dust- and particle-free to allow even seating on the array. Air bubbles trapped under the cover slip exit after several minutes at 64°C.
5. Cover slip comes off during washes.
6. Cy3 or Cy5 are scanned dry. Storage of up to 2 weeks (Dark, RT) is OK.
