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Updated: Jun 30, 2026

Interphase Fluorescence in situ Hybridization of Bone Marrow Smears of Multiple Myeloma
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Simultaneous Immunofluorescence-Based In Situ mRNA Expression and Protein Detection in Bone Marrow Biopsy Samples.

Alba Lillo Sierras1, Sandro Bräunig1, Hongzhe Li1

  • 1Division of Molecular Hematology and Stem Cell Center, Lund University, Lund, Sweden.

Bio-Protocol
|June 29, 2026
PubMed
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This study combines RNA-FISH and immunofluorescence antibody staining to simultaneously detect RNA and proteins in bone marrow biopsies. This method allows for spatial expression analysis of specific transcripts and cell markers in leukemia patients.

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:

Keywords:
Acute lymphoblastic leukemiaBone marrow fibrosisMulti-color immunofluorescence stainingRNA-scopeSpatial RNA expression analysis

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  • 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.