A more reliable and reproducible way to create fluorescently or
radioactively labeled cDNA
New Microarray Labeling Kit for Preparing cDNA Probes
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.
Arabidopsis Control mRNA
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
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.
Target DNA on Control Microarray
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 ß-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.
Specificity and Sensitivity of the Assay
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
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 ß-actin and additional target
gene-labeled probes are generated from the researcher’s test or control mRNA
in the probe-labeling reaction. The abundances of these mRNA will vary with each
tissue. The fluorescent intensities of the ß-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
Use Stratagene’s 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
Schena, M., et al. (1995) Science 270: 467-470.
Dolter, K., et al. (2000) Strategies 13: 12-14.