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

Abstract

Source: Outh-Gauer, S. et al. Chromogenic In Situ Hybridization as a Tool for HPV-Related Head and Neck Cancer Diagnosis. J. Vis. Exp. (2019)

This video describes a sequential hybridization strategy that involves hybridizing mRNA probes to specific target mRNA. Following probe hybridization, signal amplification is performed to enhance resolution via reduction of signal-to-noise ratio during RNA-CISH of histological samples.

Protocol

All procedures involving human participants have been performed in compliance with the institutional, national and international guidelines for human welfare and have been reviewed by the local institutional review board.

1. Running the Assay

NOTE: Do not let sections dry out between the incubation steps.

1. Hybridization of the HPV probe
   NOTE: Ensure the probes are prewarmed to dissolve any precipitation prior to use. For this step, instead of HPV probe, peptidyl-prolyl isomerase B (PPIB) for positive control or dihydrodipicolinate reductase (DAP....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
HybEZ Oven (110v)	Advanced Cell Diagnostics Inc.	321710
HybEZ slide rack	Advanced Cell Diagnostics Inc.	300104
RNAscope 2.5 HD Detection Reagents-BROWN	Advanced Cell Diagnostics Inc.	322310	This kit includes amplification reagents AMP1, AMP2, AMP3, AMP4, AMP5 and AMP6, and detection reagents DAB-A and DAB-B
RNAscope 3-Plex Negative Control Probe	Advanced Cell Diagnostics Inc.	320871	DAPB
RNAscope 3-Plex Positive Control Probe	Advanced Cell Diagnostics Inc.	320861	PPIB
RNAscope Probe- HPV16/18	Advanced Cell Diagnostics Inc.	311121
RNAscope Wash Buffer Reagents	Advanced Cell Diagnostics Inc.	310091	Wash Buffer 50X x4