Accurate Measurement of Cell Number-Normalized Differential Gene Expression in Cells Treated With Retinoic Acid

Area of Science:

• Genomics
• Molecular Biology
• Cancer Research

Background:

• Genome-wide gene expression analysis using RNA sequencing (RNA-seq) is crucial for understanding cellular states.
• A key assumption in bulk RNA-seq is equivalent RNA output per cell across samples, which is often violated when MYC or MYCN expression varies significantly.
• High MYC/MYCN levels in cancer cells can drastically increase overall mRNA per cell, complicating expression analysis.

Purpose of the Study:

• To develop and describe a robust RNA-seq analysis approach for MYCN-amplified neuroblastoma cells.
• To accurately normalize gene expression data on a per-cell basis, especially under conditions of varying MYCN levels.
• To enable precise assessment of differential gene expression following treatments that alter MYCN expression.

Main Methods:

• Utilized RNA sequencing (RNA-seq) on MYCN-amplified neuroblastoma cells treated with retinoic acid.
• Incorporated spiked-in RNA standards, added proportionally to the cell number in each sample before RNA extraction.
• Standardized gene expression levels to the spiked-in RNA standards for per-cell normalization.

Main Results:

• Retinoic acid treatment led to significant downregulation of MYCN expression in neuroblastoma cells.
• This MYCN reduction caused substantial changes in overall cellular RNA expression levels per cell.
• The spiked-in RNA standard method provided accurate normalization, reflecting true global expression changes.

Conclusions:

• The described protocol offers a step-by-step method for normalizing RNA-seq data on a per-cell basis.
• This approach is vital for accurate differential gene expression analysis in cells with disparate MYC or MYCN levels.
• Accurate normalization is essential for understanding treatment effects, such as retinoic acid's impact on MYCN-driven neuroblastoma.