Integration of Sigma^® TransPlex^® WTA and Complete WTA2 Kits with the Agilent Microarray Workflow

Amplification products generated by the TransPlex^® WTA and Complete WTA2 kits are suitable for microarray target for expression analyses, and can be readily integrated into existing Agilent workflows. The following modifications are required:

• The TransPlex WTA amplification product is double-stranded cDNA. Labeling, hybridization, and wash procedures are preformed using the workflow outlined in the Agilent Oligonucleotide Array-Based CGH for Genomic DNA
  Analysis ULS Labeling for Blood, Cells, Tissues or FFPE (with a High Throughput option) (Version 3.4, July, 2012).
• Fragmentation of the amplification product is not required.
• The Agilent Genomic DNA ULS™ (Universal Linkage System) Labeling Kit procedure (Agilent Cat. No. 5190-0419), allows for direct labelling of the microarray target, preferable to enzymatic incorporation of modified nucleotides during or following amplification.

Preparation of WTA Amplification Product for Labeling

1. Perform WTA amplification as described in the product bulletins for the TransPlex WTA or Complete WTA2 kits, found on the Sigma-Aldrich website.
   
   
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.    Thirty microliters is the minimum elution volume Elute with 50 ul for maximum yield.
   
   Note 2. The capacity of the GenElute PCR Cleanup filter cartridge is 10 μg, sufficient for the typical yield of a single Transplex WTA amplification reaction.
   
   Note 3. Divalent cation contamination (e.g. Mg 2⁺) will negatively affect ULS labelling efficiency.
   The GenElute PCR Cleanup kit adequately removes divalent cation contaminants and chaotropic salt contamination.
   
   
3. Determine concentration of purified amplification product using Nanodrop spectrophotometry. Use the following table (adapted from the Agilent procedure) to assure you have the appropriate DNA concentration for target labeling for the respective Agilent array formats
   
   Note 4. The DNA volume shown is required for subsequent steps in the Agilent workflow. Add nuclease-free water to bring to volume if necessary.

* Input DNA requirements and volumes are the same for both FFPE and non-FFPE samples for the 2x, 4x, and 8x arrays.
† You can use more DNA, but you will also need to use more ULS dye. Always use a ratio of 1 μL ULS dye per microgram DNA (see procedure below).

Microarray format *	DNA input amount [ng]†	Volume of DNA [μL]	Minimum DNA concentration [ng/μL]
1x microarray (non-FFPE samples)	1500	16.5	91
1x microarray (FFPE samples)	2000	16	125
2x microarray	1000	17	59
4x microarray	500	8	62.5
8x microarray	250	8	32

4. If concentration of the amplification product is required, use vacuum-centrifugation to avoid loss of amplified product.
5. Fragmentation of WTA amplification product is unnecessary.

Entry into Agilent Workflow

1. Enter Agilent Oligonucleotide Array-Based CGH for Genomic DNA Analysis ULS Labeling for Blood, Cells, Tissues or FFPE at the step “ULS Labeling.
   
   
2. Proceed without deviation from the Agilent procedure, through the following steps:
   ULS Labeling
   Removal of non-reacted ULS-Cy
   
   
3. Follow “Preparation of Labeled ‘Genomic' DNA for Hybridization” with the following modifications:
   
   Modification 1.  Do not add Cot-1 DNA to the Hybridization Master Mix.  Substitute with nuclease-free water.  (Cot-1 DNA blocking is unnecessary for transcriptome analyses.)
       
   Modification 2.  In accordance with Modification 1 above, omit the 37 °C, 30-minute incubation. (This step is included to allow the Cot-1 DNA to hybridize to highly-repeated genomic target sequences before application to the array, for the purpose of reducing non-specific target-probe interaction. Since Cot-1 DNA is not added, omit this step.)
   
   Modification 3.  Before loading hybridization samples on the arrays, incubate the samples at 95 °C for 3 minutes.  Place immediately on ice until applying to the array.
    
   Modification 4.  Hybridize for forty hours of 65 °C in all cases."

References

1. Ken Heuermann. Sr. Scientist, Life Science Division. Sigma-Aldrich Corporation, St. Louis, MO..