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.

To start hybridization, tap and flick the slides to remove any excess liquid and place them back in the slide rack. Remove prewarmed HPV probe from the oven and add approximately four drops to entirely cover each section. Cover the tray with a lid and insert it into the oven for 2 hours at 40 degrees Celsius. After completing incubation, remove the tray from the oven and remove the slide rack.

One slide at a time, quickly remove any excess liquid and place the slide in a slide rack submerged in a staining dish filled with 1x wash buffer, washing the slides for 2 minutes at room temperature with constant agitation, and repeat with fresh 1x wash buffer. Tap and flick to remove any excess liquid from the slides and place them in the slide rack again. Add approximately four drops of room temperature AMP1 per section to entirely cover each section. Cover the tray with a lid and insert it into the oven for 30 minutes at 40 degrees Celsius.

After removing the tray from the oven, remove the slide rack. Working one slide at a time, quickly remove any excess liquid and place the slide in the slide rack submerged in a staining dish filled with 1x wash buffer, washing for 2 minutes at room temperature with constant agitation, and repeat with fresh 1x wash buffer.