Identification of internal control genes for circular RNAs

Area of Science:

• Molecular Biology
• Genomics
• Biotechnology

Background:

• Accurate quantification of circular RNAs (circRNAs) using real-time quantitative PCR (RT-qPCR) is hindered by the lack of established internal control genes.
• Reference genes are crucial for normalizing expression data and ensuring the reliability of RT-qPCR results in various biological contexts.

Purpose of the Study:

• To identify and validate stable circular RNA (circRNA) candidates for use as reference genes in RT-qPCR analyses.
• To address the critical need for reliable control genes in circRNA research.

Main Methods:

• RNA sequencing (RNA-seq) was employed to identify commonly expressed circRNAs across samples.
• Stability of candidate circRNAs and traditional linear control genes (GAPDH, β-actin, 18S rRNA) was evaluated across 22 cell lines.
• Candidate circRNAs were further assessed under conditions involving linear RNA removal and treatment with Adriamycin, NH4Cl, and shikonin.

Main Results:

• Fifty circRNAs were identified as consistently expressed, with the six most stable circRNAs selected for further analysis.
• hsa_circ_0000284 (circHIPK3) and hsa_circ_0000471 (circN4BP2L2) demonstrated the highest stability among all tested genes, including linear controls.
• These two circRNAs maintained their stability even after linear RNA removal or in cells treated with Adriamycin, NH4Cl, and shikonin, exhibiting high amplification efficiencies (95-100%).

Conclusions:

• hsa_circ_0000284 and hsa_circ_0000471 are validated as highly stable and suitable reference genes for circRNA quantification via RT-qPCR.
• The superior stability of these circRNAs compared to linear RNAs suggests their potential application as reference genes for various RNA types.
• This study successfully fills the existing gap in validated reference genes for circRNA detection, significantly advancing the field.