Pixelated spatial gene expression analysis from tissue.

Area of Science:

• Molecular Biology
• Biotechnology
• Histology

Background:

• Accurate spatial localization of nucleic acids within tissue is crucial for understanding disease mechanisms.
• Existing methods for nucleic acid analysis in tissues often require complex purification steps and can compromise spatial integrity.
• There is a need for rapid, quantitative, and spatially resolved molecular profiling of histological samples.

Purpose of the Study:

• To develop and validate an on-chip technique for performing picoliter real-time reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) directly on histological tissue sections.
• To preserve the native spatial location of nucleic acid molecules during amplification.
• To enable rapid, quantitative molecular analysis of tissue samples.

Main Methods:

• Development of an on-chip system for picoliter RT-LAMP reactions.
• Direct amplification of target messenger RNA (mRNA) from unprocessed histological tissue sections.
• Demonstration using TOP2A mRNA amplification in a prostate cancer xenograft.
• Validation using mRNA fluorescence in situ hybridization (mFISH).

Main Results:

• Successful on-chip RT-LAMP amplification of TOP2A mRNA in a prostate cancer xenograft with 100 µm spatial resolution.
• Visualization of variations in amplification threshold time across the tissue, indicating spatial heterogeneity.
• Validation of on-chip RT-LAMP results by mFISH.
• Complete process turnaround time of less than 2 hours.

Conclusions:

• The developed on-chip RT-LAMP technique allows for rapid, spatially resolved, and quantitative molecular analysis of histological tissue sections without analyte purification.
• This method preserves the native spatial information of nucleic acids within the tissue.
• The technique is anticipated to be a valuable tool for biomedical researchers and clinicians, facilitating tissue analysis for diagnostics and research.