Methods and product for optimising localised or spatial detection of gene expression in a tissue sample

A method for localized or spatial detection and/or analysis of RNA in a tissue sample or a portion thereof section, comprising:

• (a) providing an object substrate on which at least one species of capture probe, comprising a capture domain, is directly or indirectly immobilized on the object substrate such that the probes are capture probe is oriented to have a free 3′ end to enable said capture probe to function as a reverse transcriptase (RT) primer,
• (b) contacting said object substrate with a tissue sample section and allowing RNA of the tissue sample section to directly hybridise to the capture probes under a set of conditions;
• (c) generating first strand cDNA molecules from the captured hybridised RNA molecules using said capture probes as RT primers and a fluorescently labelled nucleotide, thereby incorporating the fluorescently labelled nucleotide into the cDNA molecules,
• wherein said incorporation of the fluorescently labelled nucleotide is contemporaneous with said generating step;
• (d) labelling the cDNA molecules generated in step (c), wherein said labelling step may be contemporaneous with, or subsequent to, said generating step;
• (e) (d) detecting a signal from the fluorescently labelled cDNA molecules on the object substrate, thereby spatially detecting RNA of the tissue section;
• and optionally
• (f) (e) imaging the tissue sample section, wherein the tissue sample section is imaged before or after step (c), and combining the signal from the fluorescently labelled cDNA molecules with the image of the tissue section, thereby spatially detecting the RNA in the tissue section.