Source: University of Michigan
Summary: The research consortium tested nine different methods for RNA sequencing to understand and standardize the best way to sequence small RNAs. A liquid biopsy relies largely on the ability to sequence small RNA such as microRNA.
The idea of testing blood or urine to find markers that help diagnose or treat disease holds great promise. But as technology has improved to allow researchers to examine tiny fragments of RNA, one major problem has led to limited success. Different people are using different methods to sequence small RNA, and sometimes getting different results. If it keeps going on like that, it will be hard for the field to make progress. microRNA can become altered in diseases such as cancer, providing a clue to help spot disease in its earliest stages. But blood or urine contains only a tiny amount of RNA outside of cells, making it challenging to sequence. The research consortium from the University of Michigan and NIH tested nine different methods for RNA sequencing to understand and standardize the best way to sequence small RNAs. A liquid biopsy relies largely on the ability to sequence small RNA such as microRNA. The study findings were published in the journal Nature Biotechnology.
For this study, researchers prepared samples identically and sent them across the country for each of the nine labs to analyze. Each lab used multiple testing protocols to sequence four different samples, including a plasma sample and three synthetic RNA samples. Altogether, they tested nine different sequencing protocols, including four commercially available kits and five protocols developed by the labs. The combined data yielded more than 5 billion sequencing reads. Researchers found that different methods used for sequencing produced different, often inaccurate, estimates of how abundant any individual marker was. The methods developed by the consortium labs improved the accuracy of these estimates. When RNA sequencing was used to compare the relative amounts of individual microRNAs between different samples, however, all the methods produced accurate and reproducible estimates.
Study author Ryan Spengler said, “We found there was not a lot of variability if you used the same protocol across multiple labs”, “This means, if you want to coordinate a study between different labs, the key is to keep to the same protocol whatever it is. Then you can compare your results.”
More Information: Maria D. Giraldez et al, “Comprehensive multi-center assessment of accuracy, reproducibility and bias of small RNA-seq methods for quantitative miRNA profiling, Nature Biotechnology (2018). DOI: 10.1038/Nbt.4183