Duplicate hybridizations in both positive and negative dye-swap polarities were done with gDNA from a female colon carcinoma cell line, using pooled normal female gDNA as a reference sample. Each protocol was evaluated for its ability to successfully detect known chromosomal variations in the colon carcinoma HT29 cell line. The HT29 line is well-characterized with documented, well-known chromosomal aberrations.
HT29 gDNA was isolated from cultured cells with the DNeasy Tissue Kit (Qiagen(www.qiagen.com)) using the supplier’s provided protocol. DNA quantity and quality were assessed by UV/Vis spectroscopy with the ND-1000 Spectrophotometer (NanoDrop Technologies(www.nanodrop.com)) and the PicoGreen® dsDNA fluorometric assay (Invitrogen(www.invitrogen.com)), in addition to gel analysis on 0.8% agarose E-gels® (Invitrogen). Low-input (requiring 500-ng gDNA) and high-input (requiring 3-µg gDNA) protocols were performed simultaneously. The low- and high-input protocols are profiled in the Agilent “Oligonucleotide Array-based CGH for Genome DNA Analysis” guides.
Appropriate cyanine 5- and 3-labeled DNA sample pairs were combined and then mixed with 50 µL of 1 mg/mL human Cot-1 DNA (Invitrogen), 52 µL of Agilent 10X Blocking Agent, and 260 µL of Agilent 2X Hybridization Buffer in a total volume of 520 µL. Prior to hybridization, the samples were heated at 95°C for 3 minutes and then incubated for 30 minutes at 37°C. 490 µL of the labeled target solution was then hybridized to Agilent’s 244K Human Genome CGH microarrays in SureHyb hybridization chambers rotated at 20 rpm for 40 hours in a 65°C oven. After hybridization, the microarrays were washed and dried. Microarray slides were immediately scanned on the Agilent Microarray Scanner.
Data for individual features on the microarray were extracted from the scan image using Agilent Feature Extraction 9.1 Software. Output files were imported into Agilent’s CGH data-analysis software, CGH Analytics 3.4. To interpret microarray performance, CGH Analytics generates a set of quality metrics including: signal intensity, background noise, signal-to-noise ratio, reproducibility of replicate probes, and probe-to-probe log ratio noise (DLRSpread).
To assess overall CGH platform performance with reduced sample input quantities, the data from duplicate hybridizations in positive and negative polarities (dye-swaps) using female colon carcinoma gDNA pooled with normal female gDNA were analyzed with the Agilent CGH Analytics 3.4. Copy number changes representing a Chromosome 8p deletion and an 8q amplification were detected by both low and high input protocols (Figure 2).
Furthermore, a single copy amplification of Chromosome 18p as well as an 18q deletion was detected by both protocol methods. Lastly, both protocols detected a previously identified homozygous deletion at the Chromosome 16 ataxin-2 binding protein 1 (A2BP1) locus (RefSeq spanning 6.0–7.7 MB) implicated in familial neurodegenerative disease (Figure 3).
The identification and analysis of genomic variations and rearrangements is vital for the advancement of cancer and genetic disease research, as well as the diagnosis of such diseases. Our results demonstrate the effectiveness of combining aCGH-targeted probe design with protocol optimization to deliver a microarray-based CGH platform capable of precise, high-sensitivity molecular assessment across the entire human genome, even when target materials are limited.
This improved workflow can augment traditional cytogenetic and microscopy techniques, giving researchers a powerful tool for genome-wide scanning and pinpointing chromosome changes.