RNAlater-compatible protocol for nuclei isolation from radical prostatectomy prostate cancer resections, enabling...

Area of Science:

• Molecular Biology
• Genomics
• Oncology

Background:

• Prostate cancer (PC) research benefits from single-cell genomics.
• Preservation of clinical samples using RNAlater presents challenges for nuclear isolation.
• Existing methods for nucleus isolation can be complex and time-consuming.

Purpose of the Study:

• To optimize a protocol for single-nucleus isolation from RNAlater-preserved prostate cancer resections.
• To enable robust and reproducible single-nucleus RNA sequencing (snRNA-seq) on these samples.
• To expand the utility of single-cell genomics in translational cancer research.

Main Methods:

• A streamlined protocol using bead-mill homogenization and high-salt lysis for nucleus suspension.
• Elimination of the need for Fluorescence-Activated Cell Sorting (FACS) or density-gradient purification.
• Generation of high-quality snRNA-seq libraries with minimal ambient RNA contamination.

Main Results:

• Successful isolation of nuclei from RNAlater-preserved prostate cancer specimens.
• High-quality sequencing libraries with Q30 scores ≥90% were obtained.
• Identification of eight distinct cellular compartments, including luminal, stromal, and immune cells, from 3,365 nuclei.

Conclusions:

• The optimized protocol enables robust snRNA-seq from RNAlater-preserved prostate cancer samples.
• This method facilitates routine analysis of clinical specimens.
• The approach broadens the application of single-cell genomics in translational prostate cancer research.