# Protocol to validate custom-designed BaseScope probes using cell-free synthesized protein lysates and in vitro-transcribed purified mRNA

## TL;DR

This paper introduces a cost-effective protocol to validate BaseScope probes using cell-free protein lysates and purified mRNA, improving efficiency for detecting rare RNA molecules.

## Contribution

A novel validation protocol for BaseScope probes using cell-free synthesized lysates and in vitro mRNA as controls is presented.

## Key findings

- Custom BaseScope probes can be validated using cell-free protein lysates and in vitro-transcribed mRNA as positive controls.
- The protocol includes steps for slide preparation, hybridization, amplification, and detection in liquid samples.
- The method is applicable to liquid biopsies and RNA therapeutic validation.

## Abstract

BaseScope is an ultra-sensitive in situ hybridization technique specifically designed for detection of rare, short RNA molecules, particularly enabling co-detection of exon junctions in splice variants. Its use is cost intensive due to required protocol optimization for each tissue, especially when target expression is very low. Here, we present a protocol to validate functionality of custom-designed BaseScope probes by utilizing cell-free synthesized protein lysates and in vitro-transcribed purified mRNA as positive controls. We detail steps for slide preparation, probe hybridization, signal amplification, and detection.

•Steps for performing a duplex chromogenic BaseScope assay using cell-free liquid samples•Procedures for validating functionality of custom-designed BaseScope probes•Guidance on using protein lysates and in vitro-transcribed mRNA as positive controls•Workflow for applying the method to liquid biopsies and RNA therapeutic validation

Steps for performing a duplex chromogenic BaseScope assay using cell-free liquid samples

Procedures for validating functionality of custom-designed BaseScope probes

Guidance on using protein lysates and in vitro-transcribed mRNA as positive controls

Workflow for applying the method to liquid biopsies and RNA therapeutic validation

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

BaseScope is an ultra-sensitive in situ hybridization technique specifically designed for detection of rare, short RNA molecules, particularly enabling co-detection of exon junctions in splice variants. Its use is cost intensive due to required protocol optimization for each tissue, especially when target expression is very low. Here, we present a protocol to validate functionality of custom-designed BaseScope probes by utilizing cell-free synthesized protein lysates and in vitro-transcribed purified mRNA as positive controls. We detail steps for slide preparation, probe hybridization, signal amplification, and detection.