Influence of RNA extraction methods and library selection schemes on RNA-seq data

Area of Science:

• Molecular Biology
• Genomics
• Transcriptomics

Background:

• RNA sequencing is a key tool for transcriptome analysis.
• Ribosomal RNA (rRNA) depletion methods expand RNA sequencing to non-polyadenylated RNA.
• Concerns exist regarding intronic reads in rRNA depletion protocols and their nuclear origin.

Purpose of the Study:

• To evaluate the impact of RNA extraction methods and library preparation protocols on intronic read abundance and gene expression estimation.
• To investigate the origin of intronic reads in rRNA depletion sequencing.
• To provide guidelines for minimizing biases in gene expression analysis.

Main Methods:

• Comparison of two RNA extraction methods (Qiagen, TRIzol) and two library construction protocols (rRNA depletion, mRNA enrichment).
• Analysis of cytoplasmic and nuclear transcriptome fractions.
• Assessment of intronic read abundance and gene expression values.

Main Results:

• The combination of TRIzol extraction and rRNA depletion yielded the highest fraction of intronic reads, particularly when analyzing the nuclear transcriptome.
• Intronic reads largely represent unprocessed nuclear transcripts, not independent transcriptional units.
• RNA extraction methods differentially retain nuclear RNA species, affecting rRNA depletion-based sequencing.

Conclusions:

• The choice of RNA extraction method significantly influences gene expression analysis when using rRNA depletion.
• Guidelines and a double selection strategy are proposed to minimize expression biases and improve data accuracy.
• Understanding the source of intronic reads is crucial for reliable gene expression and alternative splicing analysis.