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Comparative Study
. 2004 Jan;164(1):315-23.
doi: 10.1016/S0002-9440(10)63121-5.

Detection of differentially expressed genes in an isogenic breast metastasis model using RNA arbitrarily primed-polymerase chain reaction coupled with array hybridization (RAP-array)

Derek D Sloan  1 , Ben Nicholson, Virginia Urquidi, Steve Goodison
Affiliations
Comparative Study

Detection of differentially expressed genes in an isogenic breast metastasis model using RNA arbitrarily primed-polymerase chain reaction coupled with array hybridization (RAP-array)

Derek D Sloan et al. Am J Pathol. 2004 Jan.

Abstract

To facilitate the study of the mechanisms of breast cancer metastasis we have previously characterized a pair of breast tumor cell lines that originate from the same breast tumor cell line MDA-MB-435, but which have diametrically opposite metastatic capabilities. These cell lines constitute a stable and accessible experimental system for the identification of metastasis-related genes and for the study of their role in the process of metastasis. In this study, we used a combination of RNA arbitrarily primed-polymerase chain reaction (RAP-PCR) fingerprinting and cDNA arrays (here termed "RAP-array") to identify genes differentially expressed with respect to metastatic phenotype. RAP-PCR was used to generate radioactive probes of reduced complexity for hybridization to nylon membranes containing 588 cDNAs of known identity. Single RAP-PCR fingerprint probes hybridized from 61 (10.4%) to 116 (19.7%) of the filter array targets, with a signal detection overlap of approximately 21%. A total of 344 (57%) of the 588 target genes were detected by five single RAP-PCR fingerprints. The advantage of using reduced complexity probes was highlighted by the fact that the combination of RAP probes before hybridization compromised the overall detection rate by up to 40%. Sequential application of RAP-PCR probes allowed the screening of a greater, and an alternative fraction of the transcript population than was achieved with a radiolabeled total cDNA probe. Verification by quantitative reverse transcriptase-PCR confirmed significantly increased expression of keratin 9 (>100-fold) in nonmetastatic breast tumor cells and of CD70 (fivefold) in metastatic cells. The differential expression of keratin 9 and CD70 was maintained between cells grown as primary xenografts in athymic mice. The RAP-array method enabled the detection of genes not revealed using other screening methods and that are candidates for further investigation in the context of metastatic phenotype.

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Figures

Figure 1
Figure 1
Imaging of Atlas Human Cancer cDNA arrays probed with 33P-labeled cDNA from NM-2C5 and M-4A4 cells. Each array contains 588 cDNA fragments (10 ng of DNA per spot) spotted in duplicate. Negative controls and housekeeping genes are included, and genomic DNA spots serve as orientation markers. Actual size of arrays is 80 × 120 mm. mRNA from cultured NM-2C5 and M-4A4 cells was used to generate radiolabeled cDNA probes. Arrays were exposed to a phosphorimaging system for 2 days. Images from the two blots were processed using Phoretix Array software. Quantitative data from volume reports were exported for further processing. Analysis identified three differentially expressed genes described in Table 1.
Figure 2
Figure 2
Flow chart depicting the steps involved in cDNA and RAP-PCR probe production. ss, Single-stranded; PAGE, polyacrylamide gel electrophoresis.
Figure 3
Figure 3
RAP-PCR fingerprints resolved by acrylamide gel electrophoresis. mRNA from NM-2C5 cells was reverse-transcribed and subjected to PCR using arbitrary primers B1 and B2. Single primers amplified cDNA with differing efficiencies (B1 and B2). The presence of both primers in a reaction resulted in a fingerprint of greater yield and complexity (B1+B2). MW, Molecular weight markers.
Figure 4
Figure 4
Imaging of Atlas Human Cancer cDNA arrays probed with a 33P-labeled RAP-PCR fingerprint. mRNA from cultured NM-2C5 (top) and M-4A4 cells was used to generate a radiolabeled probe, in this case Fingerprint-C. Arrays were exposed to a phosphorimaging system for 2 days. An example of a differentially expressed target gene (CTGF) is boxed and expanded (inset). Images from the two blots were processed using Phoretix Array software. Grid objects were created and used for simultaneous quantitation of all duplicate spots on each array. Background subtracted and normalized volume reports were exported for further processing. Differentially expressed genes detected are described in Table 2.
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References

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