RESUMO
Cellular mechanisms that inhibit mRNA translation by regulatory molecules involving microRNAs (miRNAs), a class of noncoding RNAs (ncRNAs), are well recognized in recent days. However, methodologies that measure these changes in cell populations lack the capabilities to observe such effects at single cell resolution. This is mostly due to the low level of transcript abundance and the heterogeneity of cell populations, together with the inability to measure transcripts and proteins at the same time. Here, we combine an in situ TaqMan PCR method with immunostaining so as to amplify low abundance transcripts in cellular compartments and image these efficiently at single cell resolution. The method offers flexibility to end-users for further fine-tuning of this optimized protocol based on the number of PCR cycles for individual genes in any cell type. After immunostaining, confocal microscopy is performed to detect the fluorescence of TaqMan probes (representing amplified transcripts/miRNA) and fluorophores tagged to antibodies (representing proteins) simultaneously. The presented technique offers an important tool to understand functional genomics as well as molecular mechanism of transcriptional and translational regulation so as to map these at single cell resolution.
Assuntos
MicroRNAs/metabolismo , RNA Mensageiro/metabolismo , Vimentina/metabolismo , Células Cultivadas , Citoplasma/metabolismo , Humanos , Microscopia Confocal , Microscopia de Fluorescência , Reação em Cadeia da Polimerase , Interferência de RNA , RNA Mensageiro/genética , Análise de Célula Única , Transcriptoma , Vimentina/genéticaRESUMO
Biology at a cellular level comes with a great amount of heterogeneity. It is now evident that even clonally propagated cells in an in vitro population do not express the same set of cellular epitopes. The vascular endothelial as well as blood cells show a very high degree of heterogeneity in expression of specific proteins. Although several methods exist for identification of genome or transcriptome from a single cell, there is still limited advancement in detection of multiple cellular antigens in a single cell. This has been mainly due to the limited availability of different antibodies. Single-cell detection methods involving the use of multiple monoclonal antibodies generated in the same species would therefore provide with an important tool for cellular detection of antigens. Here, we describe a method to assess multiple proteins in a cell using different antibodies generated in the same species.