Amplification of Representative cDNA Pools from Microscopic Amounts of Animal Tissue

Abstract

The possibility of amplifying total complementary DNA (cDNA) obtained from small amounts of biological material is not yet routinely considered, despite the fact that obtaining amounts of material suitable for direct processing by standard methods is often time-consuming and expensive and may be even impossible. Perhaps the most significant obstacle to the full appreciation of the technique is the widespread belief that polymerase chain reaction (PCR) amplification severely distorts the original cDNA profile, so that some cDNA species dramatically rise in abundance while others diminish and may even become completely lost. However, we found that there are just a few simple rules that should be followed to ensure that the amplified sample is minimally distorted and fully representative (i.e., contains all types of message originally present in RNA, even the least abundant ones). This was demonstrated in our own experiments on differential display (1) and elsewhere in application of amplified cDNA as a probe for gene profiling by array technology (2–5). According to our experience in gene hunting in various biological models, amplified cDNA can substitute for normal, nonamplified cDNA in virtually all tasks. Moreover, in PCR-based gene hunting techniques such as rapid amplification of cDNA ends (RACE) (6,7; see also Chapter 5 of this volume), subtraction (8), or differential display (9), the amplified cDNA usually outperforms the normal one, because all backgrounds are predictable and can be easily kept under control.