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.