Qiaseq mirna library qc qpcr assay kit

1.5 μl of eluate was used for assessing the quality of isolation, the absence of inhibitor for enzymatic reaction, and a group of endogenous controls by real-time polymerase chain reaction (PCR). More precisely, the kits QIAseq miRNA Library QC qPCR Assay Kit (Qiagen, Cat# 331551), miRCURY LNA miRNA PCR Assay (Qiagen, Cat# 339306-YP00203907) and miRCURY LNA SYBR Green PCR Kit (Qiagen, Cat# 339347) were used.

The QIAseq miRNA Library QC qPCR Assay Kit (Cat. no. 331551, Qiagen, Germany) was used to evaluate RNA isolation quality before small RNA library preparation and to assess NGS performance post-sequencing. The kit provides 52 Spike-Ins controls with a qPCR panel that monitors the technical quality of the whole process from RNA isolation (by evaluating the reproducibility) to sequencing data analysis (by checking the reads). This method also enables the detection of enzymatic inhibitors or nucleases and hemolysis assessment (necessary for plasma miRNA identification). In brief, the procedure started during RNA isolation with the addition of 52 QIAseq miRNA Library QC Spike-Ins to the samples. The sample evaluation was determined using qRT-PCR. For the identification of RNA isolation efficiency, the calculation of delta CT for UniSp100 (CT: 31–34 range) and UniSp101 (CT: 25–28 range) was assessed and found to be within the recommended value of around 5–7. For the inhibitor detection, the UniSp6 was measured. The value was <2 CTs between any two samples. For hemolysis, delta CT (miR-23a–miR-451a) was less than 5, ensuring high-quality samples. A value of 5–7 was considered a borderline sample. Samples with a value > 7 were not used.