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LIVER Publication-derived

Fatty Liver Disease Reference Datasets

Full SOP with Academic or a plan

Source Zhao et al., 2025 · National University of Singapore; Lund University · 10.7554/elife.109534.1.sa3

Authors: Qing Zhao, William De Nardo, Ruoyu Wang, Yi Zhong, Umur Keles, Gabrielé Sakalauskaite, Li Na Zhao, Huiyi Tay, Sonia Youhanna, Mengchao Yan, Ye Xie, Youngrae Kim, Sungdong Lee, Rachel Liyu Lim, Guoshou Teo, Pradeep Narayanaswamy, Paul R Burton, Volker M Lauschke, Hyungwon Choi, Matthew J Watt, Philipp Kaldis Duration: 4 days 7 phases Cell source: Human liver tissue (biopsy from obese individuals)

Abstract

This protocol characterizes the molecular landscape of early metabolic dysfunction-associated steatotic liver disease (MASLD) in obese individuals through integrated transcriptomic and metabolomic profiling of liver biopsies and plasma. The workflow identifies distinct molecular signatures associated with hepatic steatosis and fibrosis progression, revealing dysregulated lipid and amino acid metabolism, mitochondrial dysfunction, and novel GTPase-related gene signatures linked to fibrosis initiation.

Cell source
Human liver tissue (biopsy from obese individuals)
Application
Disease modeling; Metabolic profiling; Molecular characterization of early MASLD

Protocol overview

31 steps across 7 phases

Phase 1 · Cohort Recruitment and Sample Collection Pre-operative day
  1. a Patient Recruitment and Screening
  2. b Blood Collection
  3. c Liver Biopsy Collection
  4. d Histopathological Assessment
Phase 2 · Sample Preparation for Untargeted Metabolomics Post-collection (sample processing day)
  1. a Liver Tissue Metabolite Extraction
  2. b Plasma Metabolite Extraction
  3. c Extract Reconstitution and Preparation
Phase 3 · Untargeted Liquid Chromatography–Mass Spectrometry (LC-HRMS) Analysis Analysis phase
  1. a LC-MS System Setup and Calibration
  2. b Polar Metabolite Analysis (HILIC)
  3. c Lipid Analysis (Reverse Phase)
  4. d DDA Acquisition on QC Samples
Phase 4 · Metabolomics Data Processing and Quality Control Post-acquisition (data analysis phase)
  1. a Raw Data Conversion and Import
  2. b SWATH-MS Feature Extraction and Quantification
  3. c Quality Assurance: QC Sample Analysis
  4. d Metabolite Annotation and Semi-Quantification
Phase 5 · RNA Sequencing and Gene Expression Analysis Post-sample collection
  1. a RNA Extraction and Quality Assessment
  2. b cDNA Library Preparation
  3. c RNA Sequencing on NoveSeq Platform
  4. d Raw Sequencing Data QC and Alignment
  5. e Gene Expression Data Normalization
Phase 6 · Statistical Analysis and Integrative Omics Integration Data analysis phase
  1. a Linear Regression Analysis of Metabolite and Gene Associations
  2. b Partial Correlation Network Analysis
  3. c Identification of Steatosis-Specific and Fibrosis-Specific Genes
  4. d Identification of Progressive Fibrosis Markers
  5. e Cell-Type-of-Origin Mapping
  6. f GTPase Signaling Network Analysis
Phase 7 · 3D Liver Spheroid MASH Model and Validation Experiments Experimental validation phase (4+ weeks culture)
  1. a 3D Liver Spheroid Generation
  2. b Spheroid Maturation and Disease Induction
  3. c Elafibranor Treatment of MASH Spheroids
  4. d RNA Extraction and Gene Expression Analysis from Spheroids
  5. e LX-2 Hepatic Stellate Cell GTPase Inhibition Studies

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Attribution

This SOP was written by Organthis based on the published method in Zhao et al., 2025. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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