Probe Hybridization and Signal Amplification in RNA In Situ Hybridization: A Technique for Detecting Specific RNA Sequences in Tissue Sections

To detect a specific RNA sequence in a cell, begin by taking a tissue section on a glass slide. Overlay the tissue section with a suitable hybridization buffer containing the target RNA probes.

These probes are Z-shaped RNA molecules having a target recognition portion at one end and a tail sequence at another end. A spacer arm links these two ends.

Now, incubate the slide in a hybridization oven at an optimum temperature. Z-shaped RNA probes enter the cell and hybridize with the corresponding target mRNA sequence in pairs.

Remove the slide from the oven. Immerse the slide in a washing solution to remove any unbound probes and excess hybridization buffer.

Thereafter, add a pre-amplifier solution and incubate. The pre-amplifier molecule attaches to the tail sequence of two adjacent Z probes. This molecule does not bind to a single Z probe, ensuring specificity.

Next, add the amplifier reagent mixture and incubate. Multiple amplifier molecules hybridize to the single pre-amplifier sequence.

Finally, label the tissue section with the desired labeling probes to detect the presence of a specific RNA sequence in the cell.