Representative in Vitro cDNA Amplification From Individual Hemopoietic Cells and Colonies

Abstract

A simple procedure is described for preparing microgram amounfs of cDNA, suitable for cloning into libraries, from samples as small as a single cell. Synthesis of cDNA is primed with oligo(dT) and the resulting strands are tailed with poly(dA). The c DNA is then amplified u sing an o ligo(dT)-containing sequence to p rime the polymerase chain reaction. By limiting the size of the first cDNA strands, efficient amplification is achieved independently of size of the original mRNA transcripts. The method was applied to small numbers of hemopoietic cells of various lineages, as well as to single micromanipulated cells. The results es tablished the fidelity and specificity of t he a mplified p roduct, and showed that sensitivity was s ufficient t o d etect moderate-to-low abundance messages in a s ingle cell. Amplification o f defined RNA mixtures c onfirmed that the procedure could detect low abundance sequences at least as rare as .025%, and that relative abundance was preserved during amplification.