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Protocol cDNA-Microarray Hybridization

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2680

 

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

     

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