Self-self Hybridization As An Alternative Experiment Design to Dye Swap for Two-color Microarrays

Abstract

Dye-specific bias effects, commonly observed in the two-color microarray platform, are normally corrected using the dye swap design. This design, however, is relatively expensive and labor-intensive. We propose a self-self hybridization design as an alternative to the dye swap design. In this design, the treated and control samples are labeled with Cy5 and Cy3 (or Cy3 and Cy5), respectively, without dye swap, along with a set of self-self hybridizations on the control sample. We compare this design with the dye swap design through investigation of mouse primary hepatocytes treated with three peroxisome proliferator-activated receptoralpha (PPAR␣) agonists at three dose levels. Using Agilent's Whole Mouse Genome microarray, differentially expressed genes (DEG) were determined for both the self-self hybridization and dye swap designs. The DEG concordance between the two designs was over 80% across each dose treatment and chemical. Furthermore, 90% of DEG-associated biological pathways were in common between the designs, indicating that biological interpretations would be consistent. The reduced labor and expense for the self-self hybridization design make it an efficient substitute for the dye swap design. For example, in larger toxicogenomic studies, only about half the chips are required for the self-self hybridization design compared to that needed in the dye swap design.

Gene expression profiling of canine osteosarcoma reveals genes associated with short and long survival times

“…A common reference cRNA pool consisting of all 32 tumors was used to hybridize against each tumor on the array. Dyes were swapped as described in previous literatures [ 61 ], where equal numbers of samples within each tumor group were subjected to dye swap to avoid dye bias effect. Defective spots were flagged and normalization was carried out using the Lowess print-tip normalization technique.…”

Identification of the Schistosoma mansoni TNF-Alpha Receptor Gene and the Effect of Human TNF-Alpha on the Parasite Gene Expression Profile

“…Each sample was separately labeled with either Cy3 or Cy5 and 825 ng cRNA from each amplification were used for hybridization; in experiments with schistosomula a control sample was combined with a treated sample and hybridized; in experiments with adult worm samples Self-Self hybridizations were performed [38]. Washing and scanning procedures were according to the manufacturer's instructions using GenePix 4000B scanner (Molecular Devices, Sunnyvale, CA, USA).…”

Expression Profiling of a Heterogeneous Population of ncRNAs Employing a Mixed DNA/LNA Microarray

“…We used 5  μ g total mouse brain RNA for our initial proof of concept experiments. To exclude dye bias effects [19], AlexaFluor3 and AlexaFluor5 labeled total mouse brain RNA replicas were self-self hybridized on the custom DNA/LNA chip. Analysis of the results showed that neural miRNAs such as miR-9 and miR-9* were well detected (Figure 1(a)) in contrast to snoRNAs which were almost undetectable (Figure 1(b)).…”