PNNL-13501 - Pacific Northwest National Laboratory

Results and Accomplishments
High Throughput PCR Amplification of cDNA from
Bacterial Culture
We successfully completed the amplification of 2000
mouse cDNA by PCR. PCRs were performed in 96-wells
format directly from bacterial lysate, and products were
purified subsequently (see Figure 1).
Figure 1. PCR products derived from 2304 mouse cDNA
clones
Construction of Control RNA and DNA Fragment
Two control genes have been constructed from the
kanamycin and ampicillin genes by adding a T7 promoter
and a polyA tail to the each of the DNA fragments. A
few hundred micrograms of RNA transcripts have been
produced to be used as internal controls for balancing
signals. Control RNA was successfully labeled with Cy3
and hybridized with a testing slide with the control cDNA
spotted a hundred times (Figure 2).
Figure 2. Hybridization of Cy3-labeled control RNA
(kanamycin gene) with control PCR fragments on glass slide.
Control PCR fragments were spotted 50 times at two
concentrations on aldehyde treated slide.
46 FY 2000 Laboratory Directed Research and Development Annual Report
Fabrication of cDNA Microarray
A program has been written with software AxSys TM and
has been used to control the arrayer in spotting
approximately 2000 cDNA on a single glass slide.
Spotted cDNA was cross-linked on a chemically treated
surface. We have successfully cross-linked cDNA on
aldehyde or polylysine coated glass surface (Figure 3).
Figure 3. Visualization of immobilized 1920 unique PCR
fragments using Cy3-labeled control RNA by hybridization
at low stringency
Establishing cDNA Labeling Methods
Two cDNA labeling methods have been shown to be
effective in labeling target total RNA (Figure 4).
Reagents from Genisphere Inc. that use capturing oligos
to capture a three-dimensionally linked Cy3 or Cy5
molecules for signaling amplification seems more costeffective.
Figure 4. Analysis of gene expression profile of murine
macrophage after infected with Salmonella for 19 hours
Summary and Conclusions
The process to fabricate mouse cDNA microarrays has
been established. The ability to amplify cDNA from
bacterial culture lysate allows us to generate thousands of