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Microarray Protocol cDNA-Microarray Hybridization

Please contact Shufen Chen x4275 for any questions
 
 

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)

Probe preparation
  • 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

  •  
     

    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