Skip to main content

Table 1 Techniques available for in planta cell level expression analysis

From: Protocol: a fast and simple in situ PCR method for localising gene expression in plant tissue

Technique Type Comments References
Specific gene promoter: indicator protein fusions [i.e. beta-glucuronidase (GUS) or fluorescent proteins] 1, B Long lead times of >2 months to get stably expressed genes, homozygous gene expression, sequences other than the promoter may control gene expression, no guarantee that promoter fragment chosen is correct, not suitable for plants that cannot be transformed. [5]
Laser Capture Microdissection (LCM) and Single Cell Sampling (SiCSA) 2, A Difficulty in isolating certain cell-types (e.g. vascular cells using SiCSA), long tissue prep (~2 weeks) for LCM, need for specialised equipment [6, 7]
in situ PCR 3, B No specialised equipment required apart from vibratome, simple and fast method. No detailed protocol for plants until this manuscript, especially for agarose- embedded vibratome- sectioned in tube PCR. Can be performed on plants that are difficult to, or cannot be transformed. We do not recommend fluorescence detection of in situ PCR products due to interference with autofluorescence from plant tissue. As it is possible to do separate PCRs on adjoining tissue sections or replicate tissues from other plants we see no great advantage in multiplexing in situ PCR as it causes multiple complications to what is a robust and relatively simple technique. We do not recommend multiplexing in situ PCR due to the differential abundance of transcripts in the same cell and consequently the saturation of products, generation of non-specific products and problems with signal separation.  
in situ hybridisation (ISH) 3, B High detection threshold (10–20 copies per cell for ISH vs 1–2 copies per cell for in situ PCR), need to design a specific probe that hybridises to RNA while in situ PCR uses the same primers as qPCR, much cheaper. The View RNA Assay (Affymetrix) enables automated multiple transcript detection using fluorescence by employing the principles of ISH to detect nucleic acid targets within specific cells/cell-types. Its use is limited to abundant transcripts and laboratories equipped with and/or experienced in FFPE (formalin-fixed paraffin embedded) and frozen tissue preparation. [8]
Protoplasting of fluorescently labelled cells and single cell sorting (FACS) 4, A Can assay multiple transcripts at the same time. Potential damage responses of tissue. Needs specialised sorting flow cytometer. Limited to analysis of cells that are fluorescently labelled. Cannot give detail of all cells in which a particular gene is expressed. [9, 10]
Nuclear sorting, INTACT (isolation of nuclei tagged in specific cell types) or ribosomal binding techniques 4, A When combined with microarrays or RNAseq can assay multiple transcripts at the same time. Limited to analysis of cells that are fluorescently labelled. Cannot give detail of all cells in which a particular gene is expressed. Not easily replicated. [1113]
In situ RNA sequencing 3, B Offers the prospect of obtaining whole transcriptomes and more from single cells in tissue sections. Not yet optimised in any tissue. Has not been performed for plant tissue. [14]