Representational Fragment Amplification: Exponential Amplification of Fragmented cDNA Enables Multimillion-Fold Expression Testing

Microarray analysis, which enables the comprehensive examination of many thousands of genes in a single experiment, is a promising method for furthering understanding of disease states. Because of the large amounts of probe required, however, microarray analysis has not been possible for small excision biopsies, fine needle aspirates, and microdissected tissue samples. Linear amplification of target cDNA with T7 RNA polymerase (1) is currently the most common method for the amplification of RNA for microarray analysis and has been validated (2) and optimized(3)(4). Other linear RNA amplification strategies have been developed (5)(6), but these do not generate sufficient amounts of probe for microarray analysis. DNA fragments have been used for enriching populations (7), cloning differences (8), and subtractive screening(9). Representational fragment amplification (RFA) is a method that we have developed for global amplification of cDNA as universally primed fragments. The product of RFA is double-stranded DNA, which can be directly labeled for microarray analysis, screened for genetic variation with traditional probes, analyzed with PCR-based protocols, or stored for future analysis.To perform RNA isolation, we processed paired nondiseased and diseased cervical biopsy samples from patients diagnosed with squamous cell carcinoma of the cervix as previously described (10). The specimens were anonymized by ILS Bio or Genomics Collaborative and collected with patient consent in compliance with the company Institutional Review Boards and with the Code of Federal Regulations (CFR) 45CFR46.101B. Briefly, tissue samples frozen with liquid nitrogen were ground to a fine powder, transferred to 6-mol/L guanidine-HCL at room temperature, and homogenized by multiple passes through a syringe equipped with an 18-gauge needle. We isolated RNA with a Qiagen RNeasy Midi Kit.For RFA cDNA synthesis, we used 5 μg of RNA from nondiseased and diseased tissues as templates in the Roche cDNA Synthesis System, …