Evaluation of the Performance of Two Carbodiimide-Based Cyanine Dyes for Detecting Changes in mRNA Expression with DNA Microarrays

Abstract

Microarrays have been extensively used to investigate genome-wide expression patterns. Although this technology has been tremendously successful, several practical issues would benefit from improvements in design. Here we describe a novel, efficient labeling methodology that uses carbodiimide-linked cyanine dyes to directly chemically label cDNA derived from mouse total RNA. Using this protocol, it takes only 10 min at 70 degrees C to complete the cDNA labeling reaction. The directly labeled cDNAs can then be hybridized to 70-mer mouse oligonucleotide arrays for expression profiling studies. Microarray analyses indicate that these cDNAs are uniformly labeled and produce higher signal intensities than conventional enzymatic direct labeling methods and comparable signal intensities to those obtained by conventional indirect labeling methods. Furthermore, verification of our microarray data using a reverse transcription PCR (RT-PCR) method indicates good agreement between the two methods. Thus, we conclude that our simplified cyanine-carbodiimide labeling method, which does not rely on the incorporation of modified nucleotides, will provide a reliable, quicker, and potentially cheaper alternative to established labeling techniques for gene expression analyses.

Molecular Docking Study of the Interactions Between Cyanine Dyes And DNA

“…Likewise, these probes appeared to be particularly useful as molecular probes for biomedical studies . More specifically, cyanine dyes were effectively used i) in cell labeling, including life-cell imaging [6,7], labeling neural circuits for the visualization of the structure and function of the brain [8,19], and stem cell tracking in neurodegenerative medicine [10,11]; ii) detection of oxidative stress and reactive oxygen species [12][13][14], iii) for synthesis of the fluorescently labeled antibodies [15]; iv) in cancer research for tumor imaging in the fluorescence-guided surgery [16,17] and in photodynamic therapy [18,19]; v) for pathogen detection [20]; vi) in gene expression studies to measure the levels of specific mRNAs or miRNAs [20,21]; vii) in high-throughput screening assays to evaluate the effectiveness and toxicity of potential drug candidates [21], viii) for the detection of biomolecules and their interactions [22][23][24][25], to name only a few. However, one of the greatest potentials of cyanine dyes lies in their application in DNA research [26][27][28][29][30][31][32].…”