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.

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.

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