Integration of Sigma® GenomePlex® WGA with the Affymetrix CGH Microarray Workflow

The GenomePlex® WGA amplification product is suitable as a microarray target for expression analysis on the Affymetrix platform, and can be readily integrated into existing Affymetrix workflows for Comparative Genomic Hybridization (CGH) analysis.1 The following modification is required:

  • The GenomePlex WGA amplification product is double-stranded DNA. However, fragmentation and labeling must be performed using the Genechip Whole Transcript (WT) Double-Stranded Target Assay Kit3 (Affymetrix Cat. No. 900812) as outlined here.

 

Preparation of GenomePlex WGA Amplification Product for Labeling

1. Perform GenomePlex WGA1, WGA2, and WGA4 procedures with the following modification. 
    The example provided is taken from the GenomePlex WGA2 bulletin.

    Enter GenomePlex Kit Procedure at the step titled Fragmentation.

    Modification 1. Fragmentation is not performed at this point in the procedure.1

    1. Place 10 μL of DNA (1 ng/ μL) sample (in nuclease-free H2O) in a PCR tube or multiwall strip/plate.

    Continue with the GenomePlex Kit Procedure at the step titled Library Preparation.

    Library Preparation

    5. Add 2 μL of 1X Library Preparation Buffer to each sample.
    6. Add 1 μL of Library Stabilization Solution.
    7. Vortex thoroughly, consolidate by centrifugation, and place in thermal cycler at 95°Cfor 2 minutes.
    8. Cool the sample on ice, consolidate the sample by centrifugation, and return to ice.
    9. Add 1 μL of Library Preparation Enzyme, vortex thoroughly, and centrifuge briefly.
    10. Place sample in a thermal cycler and incubate as follows:
        16°C for 20 minutes
        24°C for 20 minutes
        37°C for 20 minutes
        75°C for 5 minutes
        4°C hold
    11. Remove samples from thermal cycler and certrifuge briefly. Samples may be amplified immediately or
          stored at -20°C for three days.

    Modification 2. During the amplification step, dUTP is incorporated for subsequent Uracil-DNA
    glycosylase (UNG) fragmentation.2

    Replace the Amplification reaction setup step with the following:

      12. Create amplificatoin mix. For each re-amplification reaction, add the following reagents to the 15μL
          reaction from step 11:
        46.9 μL of Nuclease-Free Water
        7.5 μL of 10X Amplification Master Mix (final volume = 60 μL)

        0.6 μL of dUTP (10 mM), final conc. = 80 μM
        5.0 μL of WGA DNA Polymerase

2. Purify the amplification product using the GenElute™ PCR Cleanup kit (Cat. No. NA1020),
    eluting with sterile RNase-/DNase-free water (Cat. No. W4502 or W1754).

    Note 1. Elute with < 39 μL nuclease-free water. Thirty microliters is the absolute minimum elution volume.

    Note 2. The absolute capacity of the GenElute PCR Cleanup filter cartridge is 10 μg, equivalent to the typical output
    of a single GenomePlex WGA amplification reaction.

3. If concentration of the amplification product is required, use vacuum-centrifugation to avoid loss of amplified product.

Entry into Affymetrix Workflow

Fragmentation of the WGA-amplified product requires uracil DNA glycosylase (UDG) hydrolysis of incorporated dUTP.  This is accomplished using the GeneChip® WT Protocol at the step titled "Procedure M, Fragmentation of Double-Stranded DNA".  Proceed without deviation through the end of "Proccedure N, Labeling of Fragmented Double-Stranded DNA." Labeled target is then applied to the appropriate Genechip for analysis.

Proceed without deviation starting at step 1.

Procedures M and N require the use of the GeneChip® WT Double-Stranded DNA Terminal Labeling Kit (Affymatrix Cat. No. 900182).

1. Fragment the samples using the reactions described in Table 2.11.

Table 2.11
Fragmentation of Double-Stranded DNA

Component Volume or Amount in 1 Reaction
Double-Stranded DNA 7.5 μg
10X Fragmentation Buffer 4.8 μL
UDG, 10 U/μL 1.5 μl
APE 1, 100 U/μL 2.25 μL
RNase-free Water up to 48 μL
Total Volume 48.0 μL

 

Materials

     

References

  1. Clifford Tepper, PhD., Assistant Research 2, Biochemist, UCD, Med, Biochemistry and Molecular Medicine, and Ryan Davis, Staff Research Assistant, University of Chalifornia-Davis Cancer Center, Sacramento, CA.
  2. KDM8, a H3K36me2 histone demethylase that acts in the cyclin A1 coding region to regulate cancer cell proliferation
    Datsun A. Hsia, Clifford G. Tepper, Mamata R. Pochampalli, Elaine Y. C. Hsia, Chie Izumiya, Steve B. Huerta, Michael E. Wright, Hong-Wu Chen, Hsing-Jien Kung, and Yoshihiro Izumiya PNAS, May 2010; 107: 9671 - 9676.
  3. GeneChip®  Whole Transcript (WT) Double-Stranded Target Assay Manual, P/N 702179 Rev. 3, © 2005-2006 Affymetrix Inc.

 

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