Simultaneous Immunofluorescence-Based In Situ mRNA Expression and Protein Detection in Bone Marrow Biopsy Samples

Area of Science:

• Biochemistry
• Molecular Biology
• Oncology

Background:

• Fluorescence in situ hybridization (FISH) and immunofluorescence antibody staining (IF) are established techniques for detecting nucleic acids and proteins, respectively.
• Simultaneous detection of both RNA and protein in a single sample can provide deeper biological insights.
• Formalin-fixed paraffin-embedded (FFPE) bone marrow biopsies are commonly used for diagnostics but require optimized protocols for multiplexed analysis.

Purpose of the Study:

• To develop and optimize a protocol for the simultaneous detection of RNA transcripts and proteins in FFPE human bone marrow biopsy samples.
• To enable the spatial expression analysis of specific genes (TGFB1 and PDGFA1) and cell surface markers (CD45, CD271) in the context of acute lymphoblastic leukemia (ALL).

Main Methods:

• Combined RNA-FISH and immunofluorescence antibody staining (IF) protocol.
• Utilized fluorophore-labeled probes for RNA targets and antibodies for protein targets.
• Applied the protocol to FFPE bone marrow biopsy samples from patients with ALL.

Main Results:

• Successfully demonstrated simultaneous visualization of RNA transcripts (TGFB1, PDGFA1) and proteins (CD45, CD271) in bone marrow cells.
• Established a method for in situ RNA and surface marker analysis in routine human bone marrow biopsies.
• Enabled spatial expression profiling of target molecules within hematopoietic and non-hematopoietic cells in ALL samples.

Conclusions:

• The developed protocol allows for the simultaneous detection of RNA and protein expression in FFPE bone marrow biopsies.
• This technique facilitates the study of spatial gene and protein expression in the context of hematological malignancies like ALL.
• The optimized method provides a valuable tool for comprehensive molecular and cellular analysis of bone marrow samples.