Mining, validating, and quantifying circular RNA transcriptome from total RNA as a biomarker or target

作者：Tan Ze Wang, Raj Kumar Thapa, Frank Yu, Ayesha Warsi, Kassie Rong, Pious Jose, Tahereh Kashkoulinejad-Kouhi, Phyo W. Win, Christina A. Castellani, Tina Mele, Xiufen Zheng · 发表于：Scientific Reports · 年份：2025 · DOI：10.1038/s41598-025-05652-3 · 被引用次数：3 · 研究领域：Circular RNAs in diseases、MicroRNA in disease regulation、RNA modifications and cancer

Circular RNAs (circRNAs) participate in complex diseases and have emerged as a novel biomarker and therapeutic target. It is challenging to identify circRNAs from total RNA-seq data and to validate and quantify their levels by qRT-PCR. We sought to characterize the circRNA transcriptome from RNA-seq with total RNA from septic peripheral blood mononuclear cells (PBMCs), assess/compare commonly used aligners, and optimize validation methods, considering sepsis is the leading cause of death in intensive care units (ICU) and there is no effective treatment. PBMCs were isolated from sepsis patients before and after intensive care, and RNA sequencing was conducted with total RNA. RNA-seq data for circRNAs were analyzed using four pipelines. A circRNA transcriptome has been generated, and four alignment and annotation pipelines have been evaluated. TopHat was found to be the most sensitive aligner while MapSplice was found to be the most accurate. Overall, circRNA expression in septic PBMCs were found to be more abundant and less diverse at ICU admission (ICU-AD) compared with ICU discharge (ICU-DC). There were three differentially expressed circRNAs between ICU-AD and ICU-DC. The addition of reverse primers to a reverse transcription reaction improved reproducibility and accuracy of qRT-PCR. Among these altered circRNAs, two different isoforms of circular RNA from the ASPH gene (circASPH(2,4), and circASPH(2,3,4) with the same backsplicing junction (BSJ) were identified and specifi...