The three A. thaliana mRNA are added to the probe-labeling reaction at three different concentrations, along with test or control mRNA and labeled nucleotides provided by the researcher, to generate fluorescently cDNA probes. The probes are then hybridized to the Control Microarray to determine the effectiveness of the probe labeling and the specificity and sensitivity of the hybridization reactions.
The Control Microarray is designed to assess mRNA quality to enable researchers to identify problems associated with poor quality mRNA before hybridizing the cDNA probe to an expensive microarray. The Control Microarray contains positive-control target DNA, a negative control, and additional human target DNA (Figure 1). The positive-control target DNA on the Control Microarray are A. thaliana RUBISCO activase (rca), chlorophyll a/b-binding protein, ribulose-1,5-biphosphate carboxylase/oxygenase large subunit (rbcL) genes, and the human -actin gene. Two samples containing only 3X SSC are provided as negative controls.
The Control Microarray also contains six additional human target DNA. These target DNA are expressed in different tissues at varying abundances and provide additional information regarding the quality of the labeled cDNA probe and efficiency of the hybridization.
The A. thaliana control mRNA and target DNA on the Control Microarray
can be used to determine the sensitivity of the assay. The A. thaliana-labeled
probe is generated by adding the A. thaliana mRNA provided in the
probe-labeling kit to the probe-labeling reaction. The amounts of A.
thaliana photosystem I chlorophyll a/b-binding protein, rca and rbcL