What Experimental Approaches Can Be Performed Following Transcriptome Sequencing?
Transcriptome sequencing (RNA-seq) is a powerful technology for investigating gene expression. The data generated from RNA-seq can be utilized for a wide range of downstream analyses and experimental designs, including:
Differential Gene Expression Analysis
This is one of the most commonly conducted analyses. Researchers compare transcriptomes under different conditions or treatments (e.g., disease vs. healthy, or treated vs. control) to identify genes that exhibit significant differences in expression levels between groups.
Functional Enrichment Analysis
Genes identified through differential expression analysis are often subjected to functional enrichment analysis, such as Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, to predict their potential biological functions and the processes in which they may be involved.
Transcription Factor Target Prediction
RNA-seq data can be used to infer which transcription factors are likely to regulate the differentially expressed genes, offering insights into transcriptional regulatory mechanisms.
Investigation of RNA Editing and Modifications
RNA-seq enables the study of RNA editing events, such as adenosine-to-inosine (A-to-I) editing, and can also be used to predict RNA modification sites, contributing to the understanding of post-transcriptional regulation.
Alternative Splicing Analysis
RNA-seq allows for the detection and quantification of alternative splicing isoforms, revealing transcript diversity and potential regulatory complexity at the post-transcriptional level.
Discovery of Novel Transcripts and Genes
With RNA-seq data, researchers can identify previously unannotated transcripts and potentially novel genes, expanding the current understanding of the transcriptome.
Experimental Validation
Once potential target genes or transcripts are identified through RNA-seq, follow-up validation experiments—such as reverse transcription quantitative PCR (RT-qPCR), Western blotting, or RNA interference (RNAi)—can be performed to confirm the RNA-seq findings.
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