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GenomiPhi amplification of FFPE tissue DNA for array CGH

ened wax with pipette tip.

7. Heat sample to 45 °C briefly.

8. Add 100 µl chloroform.

9. Agitate gently and centrifuge at 10,500 x g for 15 min.

10. Remove top phase (~180 µl) to fresh tube.


Amplification by GenomiPhi DNA Amplification Kit
After DNA isolation, 10 ng was amplified using GenomiPhi DNA Amplification Kit. Amplification without a heat denaturation step (formalin fixation also contributes to DNA denaturation) was completed over 18 h at 30 °C, followed by enzyme inactivation by heating at 65 °C for 10 min. Yield was determined by the PicoGreen dsDNA method. Suitability for use in Phi29 DNA polymerase�mediated amplification was evaluated by monitoring the level of inhibition in the amplification reaction. Quality of genomic DNA and amplified product were determined by conventional PCR (5) and array CGH, respectively.

Amplification by DOP-PCR
Amplification of FFPE-extracted DNA by DOP-PCR was carried out using the Wellcome Trust Sanger Institute protocol (3). The detailed method can be obtained from http://www.sanger.ac.uk. Briefly, the protocol uses three random primers (DOP1, DOP2, and DOP3) for genomic DNA amplification using a thermophilic DNA polymerase.

Labeling and microarray hybridization
To prepare amplified DNA for microarray analysis, Cy3- and Cy5-labeled probes were generated from both amplification methods.

Cy3 and Cy5 probe labeling of amplified DNA was carried out as described in the SpectralChip 2600 manual (Spectral Genomics). This protocol is outlined in greater detail in reference 3. Genomic DNA (500 ng–2 µg) was labeled using the random priming method in the presence of
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