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.
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
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:
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.