Antisense RNA amplification for target assessment of total mRNA from a single cell.

Abstract

This protocol describes how to amplify mRNA isolated from a single cell and then analyze its gene expression profile using polymerase chain reaction (PCR). Single-cell analysis is advantageous over studies of cell populations because it allows identification of a range of normal physiological states expressed by different cells of the same cell type without the confounding effects of averaging that result from measuring physiological states of cell populations. This is especially important when addressing questions of physiology in tissues, which comprises many different cell types. However, a single cell does not contain enough mRNA for all of the expressed transcripts to be detected or measured by any current molecular biology techniques. The antisense RNA (aRNA) amplification method was developed to amplify the picogram amounts of mRNA found within a single cell to microgram amounts of aRNA after three rounds of amplification. This aRNA can then easily be analyzed by microarray or next-generation sequencing. These methods allow identification of all expressed mRNA species within a single cell, including previously unknown mRNAs or those mRNAs specifically affected by a certain treatment. mRNA species of interest identified by these techniques can be further analyzed by designing primers targeting these species and performing PCR. cDNA synthesized from RNA at any stage in the aRNA amplification procedure, including material directly from collected unamplified cells, can be analyzed using PCR. Regardless of downstream applications, single-cell aRNA amplification is a powerful tool for studying single-cell physiological dynamics.