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Updated: Jun 25, 2025
Three Differential Expression Analysis Methods for RNA Sequencing: limma, EdgeR, DESeq2
Published on: September 18, 2021
Comprehensive analysis of differential long non-coding RNA and messenger RNA expression in cholelithiasis using
Yanbo Sun1, Conghui Xu1,2, Jing Luo3
1Department of Gastrointestinal Surgery, The Second Affiliated Hospital of Kunming Medical University, Kunming, China.
Background:
The etiology of gallstone disease (GSD) has not been fully elucidated. Consequently, the primary objective of this study was to scrutinize and provisionally authenticate the distinctive expression profiles of long non-coding RNAs (lncRNAs) and messenger RNAs (mRNAs) in GSD.
Methods:
RiboNucleic Acid (RNA) sequencing was used on four paired human gallbladder samples for the purpose of this study. Differentially expressed lncRNAs (DElncRNAs) and mRNAs (DEmRNAs) were identified and subjected to analysis of their biological functions. The Pearson's correlation coefficients between DElncRNAs and DEmRNAs were computed to construct a co-expression network delineating their associations. Furthermore, both cis- and trans-regulatory networks of selected lncRNAs were established and visualized. Additionally, a competing endogenous RNA (ceRNA) regulatory network was constructed. To validate the RNA-sequencing data, we performed a Quantitative Real-time Polymerase Chain Reaction (RT-qPCR) on 10 paired human gallbladder samples, assessing the expressions of the top 4 DEmRNAs and DElncRNAs in gallstone and control samples.
Results:
A total of 934 DEmRNAs and 304DElncRNAs were successfully identified. Functional enrichment analysis indicated a predominant involvement in metabolic-related biological functions. Correlation analysis revealed a strong association between the expressions of 597 DEmRNAs and 194 DElncRNAs. Subsequently, both a cis-lncRNA-mRNA and a trans-lncRNA-Transcription Factor (TF)-mRNA regulatory network were meticulously constructed. Additionally, a ceRNA network, comprising of 24 DElncRNAs, 201 DEmRNAs, and 120 predicted miRNAs, was established. Furthermore, using RT-qPCR, we observed significant upregulation of AC004692.4, HECW1-IT1, SFRP4, and COMP, while LINC01564, SLC26A3, RP1-27K12.2, and GSTA2 exhibited marked downregulation in gallstone samples. Importantly, these findings were consistent with the sequencing.
Conclusion:
We conducted a screening process to identify DElncRNAs and DEmRNAs in GSD. This approach contributes to a deeper understanding of the genetic factors involved in the etiology of gallstones.
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