Cat. No. / ID: 333022
QIAseq Targeted RNA Panels have been developed as a Sample to Insight solution for quantitative gene expression profiling using RNA-seq. These panels integrate unique molecular indices (UMIs) and single primer extension (SPE) in a simple PCR-based library preparation workflow to deliver unbiased and accurate quantification from human, mouse and rat samples.
QIAseq Targeted RNA Indices are for indexing samples for targeted RNA sequencing and primers necessary for sequencing RNA libraries generated by QIAseq Targeted RNA Panels.
Traditional RNA sequencing methods suffer from PCR duplication and amplification bias, resulting in inaccurate gene expression analysis. By introducing UMIs (unique molecular indexes) before any amplification takes place, QIAseq Targeted RNA Panels are able to eliminate this issue to deliver accurate and digital quantification of genes (see figure "Unbiased and accurate gene quantification").
A unique feature of the QIAseq Targeted RNA Panels is the set of built-in control assays. The gDNA assays control for any gDNA contamination in the RNA sample to ensure reproducible results. The housekeeping gene (HKG) assays are used to normalize data, thereby making sample-to-sample and run-to-run comparisons possible.
The QIAseq Targeted RNA Panel workflow begins with converting total RNA into cDNA (see figure "Simple procedure"). The workflow requires minimal RNA input: typically 25 ng of total RNA can be used. No enrichment or rRNA depletion steps are necessary. The molecular indexing step makes use of a gene-specific primer (GSP1) which contains a 12-base UMI in a multiplex primer panel (targeting 12–1000 genes). After the barcoding step, the uniquely tagged cDNA is bead purified to remove residual primers, and a PCR is set up with a second pool of gene-specific adapter primers (GSP2) and the RS2 primer, which primes off of a common tag on the GSP1 primers. This reaction insures that intended targets are enriched sufficiently to be represented in the final library. The number of cycles is kept to a minimum to keep PCR-induced variations in amplification to a low level (any variations are easily corrected and accounted for with the molecular barcodes). Another quick cleanup with beads is performed, and a universal PCR is run with RS2 and FS2 primers, which also adds sample-indexing barcodes to each sample. A final cleanup with beads is performed and the library is complete, and ready for quantification and sequencing.
Purchase of QIAseq targeted RNA Panels provides access to data analysis tools at QIAGEN’s GeneGlobe portal. These include read alignment and data normalization and differential gene expression at no additional costs.
A 917-plex gene panel was used to prepare a library from 10 ng of NA12878 reference DNA. All assays were designed to be intra-exon, and are thus single copy on genomic DNA. This allows an estimation of the uniformity of amplicon performance in the library preparation step (e.g., every unique tag equals one captured copy). In terms of raw assay performance, 97.5% of assays are within 20% of mean/median molecular tag counts. For cataloged panels, any assay below 20% is redesigned and replaced. UMIs (unique molecular indexes) entirely remove this variation in RNA-seq counting.