Characterizing cellular identity at one cell resolution

Abstract

Cellular processes are regulated at multiple levels in mammalian cells, including regulation at transcription, posttranscription, translation, and posttranslational levels. Most of the present techniques enable us to understand these processes either by analyzing RNAs or proteins. Very few methodologies such as combined in situ hybridization and immunocytochemistry allow us to visualize RNAs and proteins simultaneously in single cells. However, low abundance of certain transcripts (mRNAs and miRNAs) impedes the available methodologies to detect them at single-cell resolution. Here, we present a new improvised method of in situ TaqMan PCR in combination with immunostaining, which we developed to detect low abundance transcripts along with cellular proteins in mammalian cells. Cellular imaging carried out using confocal microscopy for proteins and RNAs (noncoding and messenger RNAs) can be analyzed simultaneously at single-cell resolution. The present method provides an enhanced understanding of the transcript and protein relationship at single-cell resolution and is especially useful to understand cellular populations that are highly heterogeneous.