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Related Experiment Videos

Fluorescent labelling of cRNA for microarray applications.

Peter A C 't Hoen1, Floor de Kort, G J B van Ommen

  • 1Center for Human and Clinical Genetics, Leiden University Medical Center, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands. p.a.c.hoen@lumc.nl

Nucleic Acids Research
|February 22, 2003
PubMed
Summary
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We developed an efficient aminoallyl-UTP (aa-UTP) method for fluorescently labeling complementary RNA (cRNA). This indirect labeling technique enhances gene expression profiling sensitivity on oligonucleotide microarrays compared to direct labeling.

Area of Science:

  • Molecular Biology
  • Genomics
  • Biotechnology

Background:

  • Oligonucleotide microarrays are crucial for gene expression profiling.
  • Fluorescent labeling of complementary RNA (cRNA) is essential for microarray hybridization.
  • Existing fluorescent labeling methods for cDNA are well-established, but cRNA labeling requires optimization.

Purpose of the Study:

  • To develop and evaluate an efficient fluorescent labeling protocol for cRNA.
  • To compare indirect labeling using aminoallyl-UTP (aa-UTP) with direct incorporation of fluorescent dyes.
  • To optimize the labeling efficiency for sensitive gene expression analysis.

Main Methods:

  • Development of an aminoallyl-UTP (aa-UTP) driven cRNA labeling protocol.
  • Comparison with direct incorporation of Cy-UTPs using spotted murine oligonucleotide arrays.

Related Experiment Videos

  • Analysis of labeling efficiency via spectrophotometry and fluorescent hybridisation signals.
  • Optimization of the aa-UTP:UTP ratio for optimal dye incorporation.

    • Main Results:

    • Dimethyl sulfoxide significantly enhanced labeling efficiency during coupling of aa-modified cRNA with Cy dyes.
    • Indirect labeling with aa-UTP yielded 2- to 3-fold higher labeling degrees and fluorescent signals than direct Cy-UTP incorporation.
    • An optimal labeling degree of 1 dye per 20-25 nucleotides was identified.
    • Excessive labeling led to signal quenching, particularly for Cy5, impacting fluorescence intensity.

    Conclusions:

    • The developed indirect labeling method using aa-UTP is efficient, robust, and cost-effective for fluorescently labeling cRNA.
    • This technique enables sensitive detection of gene expression profiles on oligonucleotide microarrays.
    • The findings provide a valuable tool for researchers in gene expression studies.