In situ sequencing
The in situ sequencing (ISS) facility unit provides spatially resolved gene expression data for panels of genes at subcellular resolution. The technique has been developed in the lab of Mats Nilsson who has pioneered the field of generating in situ gene expression and mutation profiles (Ke, R., et al. 2013 Nature Methods 10, 857-860).
Unique Features of ISS
- Preservation of spatial information in tissue context
- Targeted approach, using padlock probing
- Highly specific amplification tool, offering multiple levels of molecular specificity
- Multiplexing, up to few hundred transcripts per sample
- Sensitivity, tunable
- High throughput due to wide-field imaging
In situ sequencing enables localisation and quantification of more than 100 transcripts simultaneously with subcellular resolution in a single tissue section in a single experiment.
- Initial consultation and price quote (free)
- Discussion of the experimental design (including a customprobe design service)
- Training session for sample preparation (generating RCA products in the tissues)
- Detectingsignals by the barcode-sequencing reaction and imaging
- Mapping coordinates of the gene expression on the tissue section
The method has been successfully demonstrated for a variety of applications such as mapping of
- Molecularly defined cell types
- RNA editing
- Mutational heterogeneity
across different type of sections including fresh frozen, PFA-fixed and FFPE in various tissues.
- Two automated epifluorescence microscopes (Axio imager.Z2; Zeiss) equipped with multiwavelength LED light with 6 separate wavelengths.
- Automated slide stainer (Autostainer 360; LAB Vision) which can process 36 slides at one go.
Mapping 49 gene expression in adult mouse midbrain and showedmidbrain dopamine neuron diversity
Tiklová K, Björklund ÅK, Lahti L, Fiorenzano A, Nolbrant S, Gillberg L, Volakakis N, Yokota C, Hilscher MM, Hauling T, Holmström F, Joodmardi E, Nilsson M, Parmar M, Perlmann T. Nat Commun. 2019 Feb 4;10(1):581. doi: 10.1038/s41467-019-08453-1.