RNA Sequencing for Identification of Differentially Expressed Noncoding Transcripts during Adipogenic Differentiation
Anna Luan1, Kevin J Paik, Jiang Li
1Stanford and Santa Clara, Calif. From the Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Division of Plastic and Reconstructive Surgery, and the Program in Epithelial Biology, Stanford University School of Medicine; and Affymetrix, Inc.
Background:
Adipose-derived stromal cells represent a relatively abundant source of multipotent cells, with many potential applications in regenerative medicine. The present study sought to demonstrate the use of RNA sequencing in identifying differentially expressed transcripts, particularly long noncoding RNAs, associated with adipogenic differentiation to gain a clearer picture of the mechanisms responsible for directing adipose-derived stromal cell fate toward the adipogenic lineage.
Methods:
Human adipose-derived stromal cells were cultured in adipogenic differentiation media, and RNA was harvested at days 0, 1, 3, 5, and 7. Directional RNA sequencing libraries were prepared and sequenced. Paired-end reads were mapped to the human genome reference sequence hg19. Transcriptome assembly was performed and significantly differentially expressed transcripts were identified. Gene ontology term analysis was then performed to identify coding and noncoding transcripts of interest. Differential expression was verified by quantitative real-time polymerase chain reaction.
Results:
Of 2868 significantly differentially expressed transcripts identified, 207 were noncoding. Enriched gene ontology terms among up-regulated coding transcripts notably reflected differentiation toward the adipogenic lineage. Enriched gene ontology terms among down-regulated coding transcripts reflected growth arrest. Guilt-by-association analysis revealed noncoding RNA candidates with potential roles in the process of adipogenic differentiation.
Conclusions:
The precise mechanisms that guide lineage-specific differentiation in multipotent cells are not yet fully understood. Defining long noncoding RNAs associated with adipogenic differentiation allows for potential manipulation of regulatory pathways in novel ways. The authors present RNA sequencing as a powerful tool for expanding the understanding of adipose-derived stromal cells and developing novel applications within regenerative medicine.
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