What is seqfish?

seqFISH (sequential Fluorescence In Situ Hybridization)  is a technology that enables the identification of many RNA transcripts directly in single cells with their spatial context preserved (Lubeck et al. 2014, Shah et al. 2016, Frieda et al. 2017, Eng et al. 2017). To do this, transcripts are labeled with fluorescent probes in sequential rounds of hybridization to read out the temporal barcode for each transcript. The temporal barcodes can then be decoded to uniquely identify the RNA transcripts present in the sample.  RNA can be identified directly in a variety of samples from extracted RNA to tissues.

 

 

Why seqfish?

Single molecule RNA imaging ensures absolute quantitation.

With seqFISH each transcript appears as a single dot in cells and abundance is quantified by counting the number of observed dots. There is no reverse transcription or PCR amplification required, which can bias quantification in traditional RT-PCR and RNA sequencing. In addition, seqFISH can accurately detect low copy number genes, such as transcription factors and master regulators, that cannot be detected with single cell RNAseq or immunostaining.  

 
 

Single mRNA molecules appear as dots over sequential rounds of hybridization. 

 
 
 

seqFISH can profile the transcriptome.

seqFISH can multiplex from 10s to over 10,000 mRNAs (Eng et al. 2017) with single molecule imaging, enabling unbiased discovery of genes that are relevant to biological phenomena. Furthermore, highly expressed housekeeping genes such as ribosomal genes can be avoided, allowing more accurate and efficient quantitation of key regulators that are often expressed at a few copies per cell.   

 
 

10,212 genes detected and decoded. 

 
 
 

Spatial context and microenvironment are preserved in tissues.

seqFISH can profile the transcriptome directly in tissues (Shah et al. 2016, Lignell et al. 2017), detecting thousands of biomarkers in 3D in a histological slice, compared to conventional methods which can only measure 1-3 markers.  We have demonstrated seqFISH in mouse brains, human brains, human pancreas, chick embryos, flies, and worms, and many other systems.

 
 

Transcripts profiled in individual neurons in the mouse brain.