Novel method for studying mRNA phenotypes in single or small numbers of cells

TLDR

Biological processes such as growth control are governed by short‑lived, low‑copy mRNA transcripts, and the cells involved are often available only in low numbers from in vivo sources. The authors present a method that outperforms in situ hybridization, RNA blotting, and nuclease protection for studying these short‑lived, low‑copy transcripts. The method isolates RNA from one to a few thousand cells, performs reverse transcription of the whole cellular RNA, then amplifies the cDNA with a specifically primed PCR to yield fragments visualized on agarose gels by ethidium bromide staining. Using this approach, actin mRNA was detected from a single cell (<100 cRNA molecules) and differences in RNA concentrations of less than threefold were quantified; multiple mRNA species can be assayed simultaneously, and the technique—termed single‑cell mRNA phenotyping—is applicable to low‑copy growth‑factor transcripts in cultured and in vivo cells.

Abstract

Abstract Biological processes, such as growth control, are often governed by biochemical steps involving mRNA transcripts that are short‐lived and have a low copy number. Furthermore, the cells involved in these processes are often available in low numbers from in vivo sources. We now report a method that is superior to in situ hybridization, RNA blot analysis, and the nuclease protection assay for the study of short‐lived, low‐copy‐number mRNA transcripts. The method consists of a microprocedure for isolating RNA from one to a few thousand cells and two coupled enzymatic steps: reverse transcription of whole cellular RNA, followed by amplification of the cDNA by a specifically primed polymerase chain reaction to give specific cDNA fragments that can be visualized on agarose gels by ethidium bromide staining. With this method we have detected actin mRNA from a single cell, or &lt; 100 cRNA molecules, and have quantified differences in RNA concentrations of less than threefold. The reverse transcription reaction products can be divided for the polymerase chain reaction, and several mRNA species can be assayed simultaneously. Therefore, we call the method single‐cell mRNA phenotyping. This technique is applicable to the analysis of low‐copy‐number growth factor transcripts in cells in culture and in vivo.