High-throughput RNA sequencing has revolutionized how scientists study gene expression, enabling us to measure the abundance of RNA transcripts and discover new types of RNA. One such type is circular RNA (circRNA), a molecule that forms a closed loop, differing from the more common linear RNAs. While circRNAs play important roles in biology, measuring and comparing their levels across samples can be tricky due to errors in the RNA sequencing process.
When scientists prepare RNA samples for sequencing, several steps can introduce errors. These include processes like exonuclease digestion (an enzyme treatment to clean up RNA), library preparation (creating a collection of RNA molecules ready for sequencing), and the sequencing itself. These steps can interfere with accurately counting circRNAs, making it hard to compare results between different experiments, tissues, or labs.
To overcome this problem, researchers at Flinders University have developed synthetic circRNA spike-ins, or SynCRS. These are artificial circRNAs that can be added to RNA samples before the sequencing process starts. SynCRS come in different sizes and amounts, allowing scientists to calibrate their experiments and ensure that their measurements of circRNA levels are accurate. This is especially helpful when comparing data from different sources, like various tissue types or even different species.
Workflow of SynCRS use in circRNA-seq
A range of SynCRS of varying size can be utilized in this protocol. The procedure is outlined below to explain the key stages following synthesis of SynCRS. IVT, in vitro transcription.
While the protocol takes about three extra days on top of regular RNA sequencing, it’s designed to be accessible to researchers who have basic experience in molecular biology and bioinformatics.
SynCRS ensures more reliable circRNA data, leading to better comparisons across different biological samples and experiments. This technology opens up new possibilities for studying how circRNAs contribute to health, disease, and other biological processes across different organisms.
In short, SynCRS is a crucial tool for improving the accuracy of circRNA research, helping scientists uncover their roles more effectively.