Cancer is a progressive genetic disease that involves the accumulation of multiple and heterogeneous genetic and epigenetic changes. Among the 30,000 – 40,000 human genes [1
], an expanding list of genes has been shown to be involved in cancer development and progression. Recently developed technologies, including cDNA microarrays, allow cancer researchers to screen thousands of genes simultaneously to identify genes that show abnormal expression in cancers [3
Cancer researchers face a serious challenge when trying to apply these tools to clinical cancer specimens. First, cancers are often detected at a late stage in development, after multiple genetic and epigenetic changes have rendered the cancer metastatic and highly refractory even to harsh treatment. Multiple clones in the same tumor mass, which can often be distinguished by morphological features, would yield more information if they could be microdissected and analyzed separately. Second, tumor tissues invariably include a mixture of different cells, such as inflammatory cells, stromal cells, vascular cells, and others. Each type of cells may contribute to a unique aspect of the cancer phenotype and may serve as a target for treatment. Therefore, there is a need to dissect those cells for separate study. For example, it was found that tumor vascular cells have distinct gene expression profiles from those of normal vascular cells [7
], and the genes that are uniquely expressed on the surface of tumor endothelium cells can serve as targets for specific cancer therapy. Finally, a key breakthrough in cancer treatment would come from early detection and diagnosis of cancer and the study of molecular events in the early stages of cancer progression. A difficulty in studying cancer at early stages is that the tumor size is small and only limited material can be obtained through means such as fine needle aspiration. Identification of the genetic changes in such a small sample is especially challenging because only limited assays can be performed using conventional approaches. It is obvious that the full potential of genomic technologies will only be realized if they can be applied to minute amounts of biological material.
For a typical gene expression profiling experiment carried out on a glass microarray where thousands of cDNA probes are deposited in an orderly manner, RNA is first isolated from the biological material under study. The mRNA (1–5% of total RNA) is reverse transcribed to cDNA, during which process fluorescent dyes (Cy3 or Cy5) are incorporated. The labeled cDNAs are hybridized to the microarray and, after washing, the fluorescent signals are detected by a laser scanner. Current protocols typically require more than 50 μg of total RNA for consistent microarray hybridization. Several hundred milligrams of tumor tissues are often needed to obtain this much RNA, which is simply unavailable in many situations. Thus, it is crucial to develop a more sensitive and reliable procedure that requires less RNA. Several molecular biology approaches, such as T7 in vitro transcription and PCR based assays, have been attempted [8
]. However, only cursory analysis has been carried out to validate the assays, and limited confirmation experiments have been performed to evaluate the validity of the amplification results.
In this study, we performed and analyzed a set of nine microarray experiments to evaluate an amplification protocol adopted from Wang et al
]. Focusing primarily on the ability to identify differentially expressed genes, we developed the following criteria for a successful amplification protocol:
1. Because amplification may enhance the signal of genes expressed at low copy numbers, more genes should be detected by an amplification protocol than by a regular protocol.
2. Most genes detected as differentially expressed using a regular protocol should also be detected using an amplification protocol. In other words, the two protocols should reveal similar patterns of differential expression.
3. An amplification protocol should generate signal intensity profiles as reproducibly and reliably as a regular protocol.
4. Microarray results obtained from an amplification protocol should match data obtained from other molecular biology approaches such as northern blotting, western blotting and immunohistochemistry assay.
Our results showed that our amplification protocol produced reproducible, reliable microarray data that was consistent with the regular protocol. We also confirmed that our amplification protocol revealed accurate information about the differential expression of low copy number genes that failed to give sufficient signal intensities using the regular protocol. Therefore, many clinical experiments for which only a minute amount of material is available can be pursued using this protocol.