QIAseq miRNA Library QC Spike-Ins can be added directly to RNA samples before small
RNA library preparation. For more accurate ratios of spike-ins vs. endogenous miRNAs in
other sample types or when using isolated RNA samples, experimental titration of QIAseq
miRNA Library QC Spike-Ins is recommended.
The protocol consists of 5 steps:
1. Addition of 52 QIAseq miRNA Library QC Spike-Ins to the samples during RNA isolation
2. cDNA synthesis, including UniSp6 Spike-In
3. qPCR reactions
4. Sample evaluation and NGS of accepted samples
5. Evaluation of the QIAseq miRNA Library QC Spike-Ins data
Replicate RNA isolations, library preparations and sequencing runs. This graph shows the excellent reproducibility as reported by the correlation between 52 QIAseq NGS Spike-ins added during the RNA isolation. TPM = Tags per Million mapped reads.
Replicate library preparations and sequencing runs from two aliquots of kidney total RNA. This graph shows the excellent reproducibility as reported by the correlation between 52 QIAseq NGS Spike-ins added to the aliquots of total RNA. TPM = Tags per Million mapped reads.
The QIAseq miRNA Library QC PCR Array Kit uses a combination of endogenous miRNAs and synthetic RNA Spike-ins detected by LNA qPCR assays to perform a rigorous sample QC. Outlier samples and samples affected by hemolysis (e.g., samples 6 and 9) can be readily identified.
The QIAseq miRNA Library QC PCR Panel can be used with a wide range of samples, as the miRNAs on the panel are expressed in many different sample types. In this example, human serum, plasma, urine, colorectal cancer (FFPE) and mouse brain (fresh frozen) samples were analyzed.