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

Amplification products generated by the TransPlex^® WTA and Complete WTA2 kits are suitable for microarray target for expression analyses, and can be incorporated into existing Illumina workflows.

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 (Sigma Cat. No. # NA1020, GenElute PCR Cleanup Kit), eluting with sterile RNase-/DNase-free water (Sigma Cat. No’s. W4502 or W1754).
   
   Note 1. Elute with 30-50 μL nuclease-free water. Thirty microliters is the minimum elution volume, for highest
   potential concentration. Elute in 50 μL 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.
   
   
3. If necessary, adjust the concentration of the amplification product using vacuumcentrifugation to avoid loss of amplified product. Determine DNA concentration using Nanodrop spectrophotometry.
   
   
4. Fragmentation of the amplification product is not required.

Labeling Procedure

The WTA amplification product is double-stranded cDNA. Labeling is accomplished using ARP ((N-amonooxyacetyl)-N’-(D-biotinoyl) hydrazine, trifluoro-acetic acid salt) in the presence of phosphate buffered acetic acid. ARP is used for detection of nucleic acid abasic sites in neutral solutions.¹ A previous report describes the use of ARP for DNA labeling, in conjunction with dUTP incorporation during amplification and subsequent UNG digestion.² It has been shown, however, that the acidic conditions prescribed in “Illumina Solution, Application Note 2” generates sufficient abasic sites in WTA-amplified cDNA for effective labeling with ARP.^3,4

Labeling reagents:

• Labeling buffer [0.952M acetic acid (Sigma Cat. No. A6283) and 28 mM MgCl₂ (Sigma Cat. No. M1028)]
• ARP solution [11.3 mg/mL ARP (N-amonooxyacetyl)-N’-(D-biotinoyl) hydrazine,trifluoroacetic acid salt (Molecular Probes, Cat. No. A10550) in 22.4mM Phosphate (K₂HPO₄.3H₂O), Sigma Cat. No. P9666)]

Labeling:

Add 3-5 μg of WTA-amplified cDNA to labeling mix comprised of

i. 5 uL Labeling buffer [0.952M acetic acid and 28mM MgCl₂]

ii. 5 uL ARP solution [(N-amonooxyacetyl)-N’-(D-biotinoyl) hydrazine,trifluoroacetic acid salt in 22.4mM
Phosphate buffer (K₂HPO₄3H₂O]

2. Bring reaction volume to 45 μL with DNase/RNase-free water.

3. Incubate at 50 °C for 1 hour

4. Purify reaction using GenElute PCR Cleanup kit as prescribed.

5. Adjust concentrations to the following concentrations, determined by the beadchip type:

Entry into Illumina Microarray Workflow

Enter the Illumina Whole-Genome Gene Expression Direct Hybridization Assay5 at “Direct Hybridization Assay Protocols”, Section “Hybridize BeadChip”, immediately following the step titled “Quantitate RNA (Optional)”. The Illumina procedure describes the use of an amplified 'cRNA' microarray target. Modifications outlined below allow for use of the double-stranded cDNA WTA amplification product as microarray target. Follow the procedural modifications below.

1. (“Preparation”) The modified hybridization temperature is 48 °C, therefore the hybridization oven temperature should be set accordingly. Likewise, HYB and HYC tubes should be equilibrated at 48 °C for 10 minutes.
   
   
2. Prior to “Prepare RNA for Hybridization” follow “Assemble the Hyb Chambers” instructions so that BeadChip Hyb Chambers are ready for the hybridization reaction.
   
   
3. (“Prepare RNA for Hybridization”) The WTA amplification product is double-stranded cDNA. Final quantity and volume were previously adjusted in step 5 above. Heat to 95 °C for 3 minutes, then transfer quickly to 4 °C or an ice water bath.
   
   
4. Add Hyb Mix volumes (as shown in table 17 of the Illumina procedure)
   
   BeadChip Type Hyb Mix Volume
   6-Sample 20 μL
   8-Sample 10 μL
   12-Sample 10 μL
   
   
5. Promptly load reactions into the Hyb Chambers (“Load Sample”) and place at 48 °C (“Hybridize BeadChips”).
   
   
6. Proceed through the remainder of the protocol as described.