Gel-free miRNA Sample to Insight solution for differential expression analysis and novel discovery using next-generation sequencing

Features

Gel-free miRNA sequencing library prep from as little as 1 ng of total RNA

Elimination of adapter dimers and unwanted RNA species resulting in the highest fidelity and most efficient data

Integrated Unique Molecular Indices (UMIs) enable quantification of individual miRNA molecules

Includes access to the RNA-seq Analysis Portal for human, mouse and rat samples

Available with 768 unique dual indices for high-throughput sequencing on Illumina NovaSeq instruments

Product Details

Optimized reaction chemistry enables robust, miRNA-specific libraries while minimizing reaction biases and eliminating adapter dimers. Unique Molecular Indices (UMIs) tag each miRNA at an early stage, eliminating PCR and sequencing bias. Analyze stranded RNA-seq data with ease using the GeneGlobe-integrated RNA-seq Analysis Portal – an intuitive, web-based data analysis solution created for biologists and included with QIAseq Stranded RNA Library Kits.

High-throughput sequencing on Illumina NovaSeq instruments is now possible with 768 unique dual indices.

Important note: We highly recommend that data is only compared with RNA-seq libraries that use the same type of indices (single or unique dual indices) in order to ensure experimental consistency. Samples and data generated with single-end indexed libraries should only be compared with other samples and data generated with single-end indices. Samples and data generated with unique dual indices should only be compared to other samples and data generated with unique dual indices.

Performance

QIAseq miRNA sequencing enables superior data generation through an improved workflow. The standard QIAseq miRNA procedure does not require gel purification, excision and elution, providing substantial sample handling/loss and time savings.

A huge issue with miRNA sequencing workflows is the presence of adapter dimer contamination. The QIAseq miRNA Library Kit has fully optimized library process to virtually eliminate adapter dimerization, even from very low inputs of total RNA . In addition, the reaction chemistry facilitates the preparation of robust, miRNA-focused libraries while all but eliminating biases and background contaminants. Together, these benefits highlight that the QIAseq miRNA Library Kit is designed to maximize the yield of miRNA available to sequence.

Due to the growth of circulating miRNAs as potential biomarkers, the QIAseq miRNA Library Kit is optimized to map miRNA down to ultralow input levels. In addition, the kit integrates Unique Molecular Indices (UMIs) into the reverse-transcription process, enabling unbiased and accurate miRNome-wide quantification of mature miRNAs by NGS. Collectively,  QIAseq miRNA offers an unrivaled Sample to Insight solution for differential expression analysis and discovery of novel miRNAs using next-generation sequencing .

Principle

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 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 next-generation sequencing (NGS) and real-time PCR (qPCR). While NGS is the default tool for novel miRNA discovery, commercially available library preparation kits are tedious and introduce biases. As a result, qPCR has been the “go to” technology for quantification of miRNA expression, until now. QIAseq miRNA 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 UMIs to produce the most representative expression data possible.

Procedure

QIAGEN offers a true Sample to Insight workflow, from sample isolation to data analysis and interpretation. Total RNA is first extracted from biofluids (such as serum, plasma, CSF and urine), cells, fresh/frozen tissues or FFPE tissues using a miRNeasy kit. From there, miRNA sequencing libraries are prepared using the QIAseq miRNA Library Kit.

In an unbiased reaction, adapters are ligated sequentially to the 3’ and 5’ ends of miRNAs. Subsequently, universal cDNA synthesis with UMI assignment, cDNA cleanup, library amplification and library cleanup are performed. Proprietary methodology, using modified oligonucleotides, virtually eliminates the presence of adapter dimers in the sequencing library and effectively removes a major contaminant often observed during sequencing. Bead-based cleanups eliminate the majority of unwanted background noise that steals sequencing reads from a budget. The UMIs ensure that during data analysis, the sample is analyzed specifically, not amplification or sequencing artifacts. To go from sample to sequencer, the process takes only eight hours, with minimal hands-on time. Up to a maximum of 48 samples can be multiplexed.

After sequencing, “.fastq” or “.fastq.gz” file formats can be uploaded directly to the GeneGlobe Data Analysis Center for primary mapping and molecular tag counting. For well-characterized species, such as human, mouse and rat, reads are mapped to species-specific miRBase and genome databases. For poorly-characterized or novel species, read are mapped to the entire miRBase database. Secondary differential expression analysis is then performed with multiple methods for molecular tag count normalization and visualization of the resulting data.

Applications

Biofluids (such as serum, plasma, CSF and urine), cells, fresh/frozen tissue and FFPE tissues containing total and miRNA are compatible with the QIAseq miRNA Library Kit. Use the QIAseq miRNA to characterize the next circulating miRNA biomarker. Uncover miRNA signatures locked away in FFPE tissues. Completely characterize a new species. The QIAseq miRNA Library Kit has been designed to enhance yields from biofluids such as serum. In the figure Detection of miRNA, the QIAseq miRNA Library Kit shows robust detection of miRNA from serum samples. Mapped reads were then compared to adapter dimers in serum samples. QIAseq miRNA still shows superior mapping of miRNAs even with limited samples . With the QIAseq miRNA Library Kit, determine the differential expression of any known or novel miRNAs from any total RNA sample derived from any species.

QIAseq miRNA is the ultimate tool to enhance discovery and expression from large-scale projects with hundreds of samples down to the small pilot focused on a group of target miRNA. QIAseq miRNA offers an unrivaled Sample to Insight solution for differential expression analysis and discovery of novel miRNAs using next-generation sequencing.