Abstract
MicroRNAs hold great promise as biomarkers of disease. However, there are few efficient and robust methods for measuring microRNAs from low input samples. Here, we develop a high-throughput sequencing protocol that efficiently captures small RNAs while minimizing inherent biases associated with library production. The protocol is based on early barcoding such that all downstream manipulations can be performed on a pool of many samples thereby reducing reagent usage and workload. We show that the optimization of adapter concentrations along with the addition of nucleotide modifications and random nucleotides increases the efficiency of small RNA capture. We further show, using unique molecular identifiers, that stochastic capture of low input RNA rather than PCR amplification influences the biased quantitation of intermediately and lowly expressed microRNAs. Our improved method allows the processing of tens to hundreds of samples simultaneously while retaining high efficiency quantitation of microRNAs in low input samples from tissues or bodily fluids.
Highlights
Major issues plaguing small RNA library production include adapter ligation bias, adapter dimer contamination, PCR amplification bias and barcode bias[6,7,8,9]
We started with a method originally described by Tuschl and colleagues, which introduces a barcode into the 3′ adapter allowing pooling of samples after the very first step of library production[18]
By introducing unique molecular identifiers (UMIs), we show that preferential PCR amplification of specific miRNA does not appear to be a significant contributor to miRNA bias
Summary
Major issues plaguing small RNA library production include adapter ligation bias, adapter dimer contamination, PCR amplification bias and barcode bias[6,7,8,9]. We started with a method originally described by Tuschl and colleagues, which introduces a barcode into the 3′ adapter allowing pooling of samples after the very first step of library production[18]. This many-to-one sample handling strategy allows for the processing of dozens to even hundreds of samples simultaneously. Stochastic capture of miRNAs at very low input concentrations requiring high PCR cycle numbers does lead to poor and irreproducible calling of miRNA expression levels, especially of intermediately and lowly expressed miRNAs. Together, our optimized high-throughput method of library production results in the highly efficient and reproducible capture of small RNAs for downstream sequencing
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
