Dual-modality Molecular Cartography: Integrating Multiplex mRNA Detection with Protein Imaging Mass Cytometry

Sammy Ferri-Borgogno; Danielle L. Stolley; Basant T. Gamal; Christopher D. Pacheco; Ivo Veletic; Akshay Basi; Duncan H. Mak; Madeline K. McAllister; Angeliquie J. Lin; Javier A. Gomez; Anna K. Casasent; Jared K. Burks

doi:10.3791/68616

Cartografia molecolare a doppia modalità: integrazione del rilevamento multiplex dell'mRNA con la...

JoVE Journal
Cancer Research

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JoVE Journal Cancer Research

Dual-modality Molecular Cartography: Integrating Multiplex mRNA Detection with Protein Imaging Mass Cytometry

Cartografia molecolare a doppia modalità: integrazione del rilevamento multiplex dell'mRNA con la citometria di massa per imaging proteico

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06:51 min

November 14, 2025

DOI: 10.3791/68616-v

Sammy Ferri-Borgogno¹, Danielle L. Stolley², Basant T. Gamal¹, Christopher D. Pacheco², Ivo Veletic³, Akshay Basi³, Duncan H. Mak², Madeline K. McAllister², Angeliquie J. Lin², Javier A. Gomez³, Anna K. Casasent², Jared K. Burks²

¹Department of Gynecologic Oncology and Reproductive Medicine,The University of Texas MD Anderson Cancer Center, ²Department of Hematopoietic Biology & Malignancies,The University of Texas MD Anderson Cancer Center, ³Department of Leukemia,The University of Texas MD Anderson Cancer Center

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Overview

This protocol details a methodology for multiplex mRNA detection with protein imaging mass cytometry in formalin fixed paraffin embedded tissue sections. The focus is on understanding the molecular mechanisms of ovarian cancer to identify new therapeutic targets and biomarkers.

Key Study Components

Area of Science

Neuroscience
Biology
Oncology

Background

Understanding ovarian cancer at a molecular level.
Challenges in detecting lower abundance proteins.
Need for antibody validation and titration.
Complexity of pixel classification during analysis.

Purpose of Study

To find new therapeutic targets for ovarian cancer.
To identify biomarkers that predict patient survival.
To improve methodologies for protein detection in tissue samples.

Methods Used

Preparation of FFPE slides.
Use of humidity control trays for hybridization.
Application of RNA-ISH Amplifier 3 reagent.
Hybridization at controlled temperature.

Main Results

Successful detection of multiplex mRNA and proteins.
Improved methodology for analyzing tissue sections.
Insights into molecular mechanisms of ovarian cancer.
Potential for identifying new biomarkers.

Conclusions

The protocol enhances the understanding of ovarian cancer biology.
It addresses challenges in protein detection.
It lays groundwork for future therapeutic developments.

Frequently Asked Questions

What is the main focus of this study?

The study focuses on understanding the molecular mechanisms of ovarian cancer.

What are the challenges addressed in this protocol?

Challenges include detecting lower abundance proteins and the labor of antibody validation.

What methodology is detailed in the protocol?

The protocol details multiplex mRNA detection with protein imaging mass cytometry.

How are FFPE slides prepared?

Excess liquid is removed from the slides before hybridization.

What is the significance of this research?

It aims to find new therapeutic targets and biomarkers for ovarian cancer.

Questo protocollo descrive in dettaglio una metodologia per la rilevazione multiplex dell'mRNA con citometria di massa per imaging proteico in sezioni di tessuto incluse in formalina fissate con paraffina.

Ci concentriamo su una biologia speciale per comprendere meglio i meccanismi molecolari del cancro ovarico, nella speranza di trovare nuovi bersagli terapeutici e biomarcatori per prevedere la sopravvivenza del paziente. Una delle sfide attuali dell'IMC include la dissezione di proteine a minore abbondanza, il lavoro di validazione e titolazione degli anticorpi, nonché la classificazione dei pixel complessa e lunga durante l'analisi. Inizia rimuovendo il liquido in eccesso dalle vetrine preparate in FFPE.

Tira fuori la teglia per il controllo dell'umidità dal forno e metti il portalami nella teglia. Poi, si utilizza accuratamente l'amplificatore RNA-ISH 3 e aggiungi abbastanza reagente da coprire completamente ogni sezione di tessuto sulle vetrine. Chiudi la teglia e inseriscila nel forno di ibridazione per 30 minuti a 40 gradi Celsius.

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