Protocol cDNAMicroarray Hybridization

RNA preparation

We recommend the following protocols for extracting total RNA:

cultured cells: Quiagen RNeasy Kit Trizol (Gibco) tissues: Quiagen RNeasy Kit modified LiCl extraction protocol (Auffray and Rougeon, Eur. J. Biochem. 107:303-314, 1980)

RNA precipitation (total RNA 100 - 150 µg)

• 1/10 vol 3M NaOAc (ph 5.2)
• 2.5 vol 95% ethanol
• mix
• store at -70 °C for 20 min (or -20 °C for 1 h or O/N)
• spin at max. rpm for15'
• discard supernatent
• add 1x vol 75% ethanol
• discard supernatent
• air dry pellet (make sure ALL ethanol is evaporated)
• resuspend in 17 µl DEPC-H2O
• place on ice

Probe preparation

• Anneal RNA
  • start with 17 µl of resuspended RNA per probe
  • Add 2 µl oligo dT (12-18)
  • Final Volume: 19 µl (per tube)
  • incubate for 3-5 min at 65 °C
  • place on ice

• Make fluorescent cDNA probe

• 8 µl 5x first strand buffer
• 4 µl 0.1m DTT
• 4 µl 10x low dT dNTP
• 4 µl Cy3 or Cy5 dUTP
• 1 µl RNAsin
• Final Volume: 21 µl (per tube)
• Add annealed RNA to flourescent mix
• Final Volume: 40 µl (per tube)
• Add 2 µl Superscript-RT (Gibco)
• Incubate at 42 °C for one hour
• add 2 µl Superscript-RT
• Incubate at 42 °C for another hour
• Heat at 94 °C for 2-3 min

• Clean cDNA from RNA

• Add 44 µl H2O to each tube
• 10 µl 10x RNAse one buffer
• 2 µl RNAse one
• Incubate at 37 °C for 10 min
• Heat at 94 °C for 2-3 min
• Combine both probes and add 30 µl of Strata Clean Resin
• Mix and incubate RT for 1-2 min, pellet resin by brief centrifugation, save supernate.
• Wash pellet with 100 µl of water, pellet and combine supernatents

• Concentrate probes

• Add to micron YM 50 column
• Add 200 µl H2O
• Mark tube & column for orientation
• Centrifuge at RT for 8-10' at 10,000-12,500g
• Wash 3x with 400 µl H2O; discard flow through
• Invert column, place into new tube and spin 15 sec at 1000g to collect probe
• Check volume and adjust to 19.5 µl

• Prehybridization of probe

• Add 40.5 µl hybridization solution to19.5 ul probe
• add 1µl blocking solution
• total 60ul
• Heat at 94 °C for 1 min
• Centrifuge at 13,000 rpm for 5' and transfer supernatant to clean tube
• Prehybridize probe at 50 °C for one hour

Slide prep (while preparing probe)

Mark slide with a pencil

Vapor moisturize (array down) over boiling water

Quickly place in Stratalinker (DNA array up)

250 mJ (setting 2500) silane slides

Remoisten over steam

Heat snap on hot plate 3-5 seconds

Rinse slide in 0.1% SDS 10-20 secs

Rinse slide in ddH2O x 10-20 secs

Boil in water bath at 95 °C for 3-5 min

Dunk in ethanol

Spin at 1000 rpm for 4 min to remove excess ethanol (array faces out)

Prehybridization of array

Place 60 µl prehybridization solution on top of array (use pre-marked dummy slide for array orientation)

Place coverslip over array

Add 10 µl dd H2O in each corner of chamber to maintain humidity

Place slide in hybridization chamber and incubate for 1 hour in a 50 °C water bath

Hybridization

Remove cover slide by dipping slide in water

Spin at 1000 rpm for 5-10 min to remove water

Add 60 µl hybridization solution over array

Place new coverslip on array

Place in hybridization chamber

Hybridize O/N in a 50°C water bath

Wash array

Place slide in 50 ml tube with 2x SSC/ 0.1% SDS

Shake gently so that coverslip falls off

Place slide in slide holder/glass dish with several hundred mls 0.2x SSC/ 0.1% SDS

Shake slide for 10-15 minutes

Dip slide in 0.2x SSC to remove SDS

Place slide in holder with several hundred mls 0.2x SSC and wash for 10-15 minutes

(you may repeat last wash step with less SSC)

Place slide in 50 ml tube and spin for 5 minutes at 1000 rpm to dry slide (array facing outwards)

KEEP IN DARK until slide is scanned

• Scan

Solutions

Filter all solutions that come in contact with slide except blocking solution

low dTTP/dNTP solution

25 µl dGTP 25 µl dATP 25 µl dCTP 10 µl dTTP 415 µl DEPC H20 500 µl total

Prehybridization solution (Available from facility)

3.5 ml formamide 2 ml 20x SSPE 0.5 ml 10% SDS 0.5 ml 50x Denhardt's 0.2 ml ss salmon sperm DNA 3.3 ml ddH20 10 ml total

Hybridization solution (Available from facility)

700 µl formamide (35%) 50 µl 20% SDS (0.5%) 100 µl 50x Denhardt's (2.5x) 400 µl 20x SSPE (4x) 1.25 ml total

Blocking solution (20x stock)

40 µl polydA (1ug/ul) 8 µl tRNA (10ug/ul) 200 µl human/mouse Cot1 DNA (1ug/ul) 240 µl total ethanol precipitate the combined reagents Resuspend in 20 µl filtered ddH2O