A more reliable and reproducible way to create fluorescently or radioactively labeled cDNA
David T. Wong Heidi Davis Darryl Garrison Terry
Payette Matt Lundy
Kim Kuplent Mary A. Buchanan Joseph A. Sorge Rebecca L. Mullinax
Travis Ward Andrew Firmin
Nucleic acid microrrays, which contain hundreds or thousands of genes in a single format, are increasingly being used to identify differentially expressed genes. In this process, test and control mRNA are converted into cDNA probes in a reverse transcription reaction that incorporates labeled nucleotides. The test and control cDNA probes are then hybridized to arrays, the unhybridized probe is removed, and the hybridization is detected. Differences in the levels of hybridization signals of specific genes on the array correlate with differences in the abundance of those genes in the test and control mRNA used to prepare the probes.
We have developed the Microarray Labeling KitPP for use with gene microarrays1 to increase the reliability and reproducibility of identifying differentially expressed genes. The kit includes several control mRNA and two Control Microarrays designed to assess the mRNA quality and the sensitivity and specificity of the assay.
The Microarray Labeling Kit includes three artificially generated mRNA from Arabidopsis thaliana genes. They are A. thaliana RUBISCO activase (rca), chlorophyll a/b-binding protein, and ribulose-1,5-biphosphate carboxylase/oxygenase large subunit (rbcL) genes. These genes were selected because they are involved in plant-specific processes and do not have known human homologues. In addition, they do not hybridize to membrane-arrayed human cDNA from 29 different tissues. Therefore, cDNA probes generated from these genes are unlikely to hybridize to human genes.
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 mRNA added to the probe-labeling reaction correspond to mRNA of high, medium, and low abundances, respectively. The relative fluorescent intensities of the A. thaliana cDNA probes when hybridized to the corresponding A. thaliana target DNA on the Control Microarray can therefore be used to determine the sensitivity of the assay (Figure 1).
The 3X SSC negative controls can be used to determine the specificity of the assay because the labeled probe should not hybridize to the spotted 3X SSC.
The quality of the RNA is critical to successful probe preparation. The presence of contaminants, such as carbohydrates, can increase background fluorescence following hybridization. The guanidinium isothiocyanate method used in the StrataPrep total RNA miniprep kit2 has been used successfully in this application.
The human -actin and additional target gene-labeled probes are generated from the researchers test or control mRNA in the probe-labeling reaction. The abundances of these mRNA will vary with each tissue. The fluorescent intensities of the -actin and other human gene cDNA probes when hybridized to the corresponding target DNA on the Control Microarray can therefore be used to determine the quality of the researchers mRNA.
Use Stratagenes Microarray Labeling Kit to prepare reliable cDNA probes for hybridization to gene microarrays. The Arabidopsis control mRNA and Control Microarray included in the kit allow problems associated with poor-quality mRNA to be identified prior to hybridizing the probe to an expensive microarray and to determine the sensitivity and specificity of the hybridization.
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