A subscription to JoVE is required to view this content. Sign in or start your free trial.
Circular RNAs (circRNAs) are non-coding RNAs that may have roles in transcriptional regulation and mediating interactions between proteins. Following assessment of different parameters for construction of circRNA sequencing libraries, a protocol was compiled utilizing stranded total RNA library preparation with RNase R pre-treatment and is presented here.
Circular RNAs (circRNAs) are a class of non-coding RNAs involved in functions including micro-RNA (miRNA) regulation, mediation of protein-protein interactions, and regulation of parental gene transcription. In classical next generation RNA sequencing (RNA-seq), circRNAs are typically overlooked as a result of poly-A selection during construction of mRNA libraries, or are found at very low abundance, and are therefore difficult to isolate and detect. Here, a circRNA library construction protocol was optimized by comparing library preparation kits, pre-treatment options and various total RNA input amounts. Two commercially available whole transcriptome library preparation kits, with and without RNase R pre-treatment, and using variable amounts of total RNA input (1 to 4 µg), were tested. Lastly, multiple tissue types; including liver, lung, lymph node, and pancreas; as well as multiple brain regions; including the cerebellum, inferior parietal lobe, middle temporal gyrus, occipital cortex, and superior frontal gyrus; were compared to evaluate circRNA abundance across tissue types. Analysis of the generated RNA-seq data using six different circRNA detection tools (find_circ, CIRI, Mapsplice, KNIFE, DCC, and CIRCexplorer) revealed that a stranded total RNA library preparation kit with RNase R pre-treatment and 4 µg RNA input is the optimal method for identifying the highest relative number of circRNAs. Consistent with previous findings, the highest enrichment of circRNAs was observed in brain tissues compared to other tissue types.
Circular RNAs (CircRNAs) are endogenous, non-coding RNAs that have gained attention given their pervasive expression in the eukaryotic transcriptome1,2,3. They are formed when exons back-splice to each other and hence were initially considered to be splicing artifacts4,5. However, recent studies have demonstrated that circRNAs exhibit cell type, tissue, and developmental stage specific expression3,6 and are evolutionarily conserved2
This research has been performed in compliance with all institutional, national and international guidelines for human welfare. Brain tissues were obtained from the Banner Sun Health Research Institute Brain and Body Donation Program in Sun City, AZ. The operations of the Brain and Body Donation Program are approved by the Western Institutional Review Board (WIRB protocol #20120821). All subjects or their legal representatives signed the informed consent. Commercial (non-brain) biospecimens were purchased from Proteogenex.
1. RNase R Treatment
NOTE: In the following steps, the reaction volume is ad....
Data generated using a commercially available universal control RNA (UC) and using two library preparation kits, both of which include a ribo-depletion step in their protocols, was first assessed. Using an analytical workflow (Data analysis workflow, section 4), overall, a higher number of circRNAs was detected in the TruSeq datasets compared to the Kapa ones (Figure 1). Although the ribosomal RNA (rRNA) percentages were below 5% in datasets from both kits for lower input amounts (1, 2 ug), .......
In this study, two commercially available library preparation kits, pre-treatment options, and input RNA amounts were tested in order to optimize a circRNA enrichment protocol for construction of circRNA sequencing libraries. Based on this study’s assessments, a number of key aspects and critical steps in creating circRNA sequencing libraries are apparent. Our evaluation confirms the utility of RNase R pre-treatment, as reflected by the increased number of circRNAs detected. Overall, a higher diversity of circRNAs .......
The authors have nothing to disclose.
We are grateful to the Banner Sun Health Research Institute Brain and Body Donation Program (BBDP) of Sun City, Arizona for the provision of human brain tissues. The BBDP has been supported by the National Institute of Neurological Disorders and Stroke (U24 NS072026 National Brain and Tissue Resource for Parkinson's Disease and Related Disorders), the National Institute on Aging (P30AG19610 Arizona Alzheimer's Disease Core Center), the Arizona Department of Health Services (contract 211002, Arizona Alzheimer's Research Center), the Arizona Biomedical Research Commission (contracts 4001, 0011, 05-901 and 1001 to the Arizona Parkinson's Disease Consortium) and the M....
Name | Company | Catalog Number | Comments |
1000 µL pipette tips | Rainin | GP-L1000F | |
20 µL pipette tips | Rainin | SR L 10F | |
200 µL pipette tips | Rainin | SR L 200F | |
2200 TapeStation Accessories (foil covers) | Agilent Technologies | 5067-5154 | |
2200 TapeStation Accessories (tips) | Agilent Technologies | 5067-5153 | |
Adhesive Film for Microplates | VWR | 60941-064 | |
AMPure XP Beads 450 mL | Beckman Coulter | A63882 | PCR purification |
Eppendorf twin.tec 96-Well PCR Plates | VWR | 951020401 | |
High Sensitivity D1000 reagents | Agilent Technologies | 5067-5585 | |
High Sensitivity D1000 ScreenTape | Agilent Technologies | 5067-5584 | |
HiSeq 2500 Sequencing System | Illumina | SY-401-2501 | |
HiSeq 3000/4000 PE Cluster Kit | Illumina | PE-410-1001 | |
HiSeq 3000/4000 SBS Kit (150 cycles) | Illumina | FC-410-1002 | |
HiSeq 4000 Sequencing System | Illumina | SY-401-4001 | |
HiSeq PE PE Rapid Cluster Kit v2 | Illumina | PE-402-4002 | |
HiSeq Rapid SBS Kit v2 (50 cycle) | Illumina | FC-402-4022 | |
Kapa Total RNA Kit | Roche | KK8400 | |
Molecular biology grade ethanol | Fisher Scientific | BP28184 | |
Qubit Assay Tubes | Supply Center by Thermo Fischer | Q32856 | |
Qubit dsDNA High Sense Assay Kit | Supply Center by Thermo Fischer | Q32854 | |
RNA cleanup and concentrator - 5 | Zymo | RCC-100 | Contains purification columns, collection tubes |
RNAClean XP beads | Beckman Coulter Genomics | RNA Cleanup beads | |
Rnase R | Lucigen | RNR07250 | |
SuperScript II Reverse Transcriptase 10,000 units | ThermoFisher (LifeTech) | 18064014 | |
TapeStation 2200 | Agilent Technologies | Nucleic Acid analyzer | |
TElowE | VWR | 10128-588 | |
TruSeq Stranded Total RNA Library Prep Kit | Illumina | 20020596 | Kit used in section 3 |
Two-Compartment Divided Tray | VWR | 3054-1004 | |
UltraPure Water | Supply Center by Thermo Fischer | 10977-015 | |
Universal control RNA | Agilent | 740000 |
Request permission to reuse the text or figures of this JoVE article
Request PermissionThis article has been published
Video Coming Soon
Copyright © 2025 MyJoVE Corporation. All rights reserved