Kreapure columns

Human Genome CGH Microarray 244A slides (Agilent Technologies, CA, USA) were used for FFPE extracted DNA samples. We followed the protocol described in [30 (link), 31 (link)]. A total of 2.5 μg sex matched control DNA (Promega, WI, USA) was fragmented by sonication. The FFPE DNA was fragmented only if there was any large molecular weight DNA. About 500 ng of the fragmented samples were then run on 1.5% agarose gel for 1 hour to check the extent of fragmentation of the DNA. Once fragmentation was deemed appropriate, 2 μg of the control DNA was labeled with Cy3 (Agilent technologies, CA, USA) and 2μg of FFPE DNA with Cy5 (Agilent technologies, CA, USA) for 30 minutes at 85°C. After labeling the DNA was purified using KREA pure columns (Agilent technologies, CA, USA). The samples were then measured on a Nanodrop and Degree of Labeling (DOL) was calculated according to the following formula;
$\mathrm{D}\mathrm{e}\mathrm{g}\mathrm{r}\mathrm{e}\mathrm{e}\mathrm{o}\mathrm{f}\mathrm{L}\mathrm{a}\mathrm{b}\mathrm{e}\mathrm{l}\mathrm{i}\mathrm{n}\mathrm{g}=\left(340\mathrm{x}\mathrm{p}\mathrm{m}\mathrm{o}\mathrm{l}/\mathrm{\mu }\mathrm{l}\mathrm{o}\mathrm{f}\mathrm{d}\mathrm{y}\mathrm{e}\right)/\left(\mathrm{n}\mathrm{g}/\mathrm{l}\mathrm{\mu }\mathrm{o}\mathrm{f}\mathrm{G}\mathrm{e}\mathrm{n}\mathrm{o}\mathrm{m}\mathrm{i}\mathrm{c}\mathrm{D}\mathrm{N}\mathrm{A}\mathrm{x}1000\right)$
The samples were hybridized onto microarray slides only if the DOL was between 1.5–2.5%.

Genomic DNA (500 ng) from the same batch of tumour DNA as used in methylation profiling was analysed using the Agilent oligonucleotide array-bases CGH (4x microarray) following the manufacturer’s instructions (Agilent Technologies, Santa Clara, CA). In brief, genomic DNA of samples and female reference DNA (a normal control DNA) (Promega, Madison, WI) were first fragmented for 30 s at 95 °C and 30 min at 95 °C respectively, and then the reference and sample DNA were labeled with ULS-Cy3 and ULS-Cy5 dye with the ratio 1 μL per 1 μg DNA, respectively. The non-reacted Cy-ULS dyes were removed using Agilent KREApure columns to reduce possible background noise for array screening. Optimal Cy5 degree of labeling (range between 0.75 % and 2.5 %) with a Cy3 minus Cy5 range between 1 % and 2 % were used as a quality control guideline for sample labeling before hybridizing to the array. Samples and references DNA were hybridized on to the microarray at 65 °C for 40 h, and then washed and scanned. aCGH result analyses was performed using the Partek® Genomics Suite™ version 6.03 (Partek Inc., St. Louis, MO).