Small RNA-seq, including miRNA

Mature miRNAs are naturally occurring, 22-nucleotide, non-coding RNAs that mediate posttranscriptional gene regulation. Alterations in miRNA can be correlated with gene expression changes in development, differentiation, signal transduction, infection, aging and disease. Continually growing evidence associates circulating miRNA expression with both normal and disease biology as miRNAs are expressed in virtually all biofluids, including serum, plasma, cerebrospinal fluid (CSF) and urine. Specifically with cancers, numerous studies and reviews have associated the presence of various miRNAs with cancer cell proliferation, resistance to apoptosis, invasiveness and differentiation.

Quantification of miRNA expression can be performed using a variety of technologies, including NGS and real-time PCR (qPCR). While NGS is the default tool for novel miRNA discovery, commercially available library preparation kits introduce biases and involve tedious procedures. As a result, qPCR has been the most commonly used technology for quantification of miRNA expression – until now. The QIAseq miRNA Library Kit defines a new generation in small RNA sequencing products and includes several distinct features not found in other sequencing kits. With the QIAseq miRNA Library Kit, the power of NGS has been combined with single molecule quantification from Unique Molecular Indices (UMI) to generate the most representative expression data possible. In recent years, NGS has emerged as a highly advanced research tool for both high-throughput miRNA expression analysis and novel miRNA discovery. The QIAseq miRNA Library Kit procedure does not require gel purification, excision and elution, which substantially reduces the hands-on time required and noticeably shortens the length of the entire workflow. Proprietary methodology utilizing modified oligonucleotides efficiently prevents adapter dimerization in the sequencing library and the highly optimized reaction chemistry virtually eliminates biases and background contaminants, facilitating the preparation of robust, miRNA-specific libraries. The kit also integrates UMI into the reverse transcription process, enabling unbiased and accurate miRNome-wide quantification of mature miRNAs by NGS. Should a library fail pre-sequencing quality control (QC), in-line controls are included in the library generation procedure to allow the use of real-time PCR for fast and efficient troubleshooting. Both primary and secondary data analysis solutions have been developed to facilitate rapid and robust UMI counting, miRNA mapping and differential expression analysis. Overall, the QIAseq miRNA Library Kit offers an unrivaled Sample to Insight solution for differential expression analysis and discovery of novel miRNAs from cells, tissues and biofluids using integrated UMI technology. It also enables precise NGS of mature miRNAs on Illumina NGS instruments. The required amount of template for a single QIAseq miRNA sequencing reaction can range from 500 ng to as little as 1 ng of purified total RNA.