Different RT reactions using 40 ng heart and liver total RNA were profiled on the miRCURY LNA miRNome Human PCR Panel I and II on different days. The correlation between raw Cq values from all miRNAs with signals below 35 Cq values is shown (total of 297 data points).
Raw Cq values from two separate RT reactions (RT1 and RT2) on total RNA purified from 65 µl serum are shown. A total of 730 miRNAs were profiled. Only miRNAs with Cq values below 35 have been included (133 data points).
Aliquots of the same heart and liver total RNA were expression profiled using miRCURY LNA miRNA PCR Panels in two different locations (on separate days by different operators) using different Roche LightCycler 480 Real-Time PCR instruments. The correlation between average Cq values (triplicate RT reactions) from all miRNAs with signals below 35 Cq values (total of 297 datapoints) is shown.
Specificity was assessed using 8 pools each containing at least 80 different synthetic miRNAs. Closely related miRNAs are placed in different pools. This example shows results from the hsa-miR-1 assay. The miRCURY LNA miRNA PCR System performs perfectly with a high signal in pool 1, which contains the hsa-miR-1 synthetic miRNA target, and only a borderline signal in pool 7, which contains the most closely related miRNA (hsa-miR-206). The competitor platform uses ligation-based cDNA synthesis and only one miRNA-specific probe. Competitor T shows a lack of specificity. The highest signal for Competitor T is obtained with pool 1. However, non-specific false-positive signals are obtained from all other pools in the absence of hsa-miR-1 template, within a Cq range that would be interpreted as real signals.
Real-time PCR was performed using triplicate RT reactions on total RNA purified from serum. Average Cq (quantification cycle) values from the miRCURY LNA miRNome Human PCR panel I are shown (368 miRNAs). Over 120 miRNAs showed robust expression with Cq values below 35. hsa-miR-16, which is known to be highly expressed in serum/plasma, and hsa-let-7a are indicated in red. The U6 reference gene is indicated in orange.
Real-time PCR was performed using triplicate RT reactions per sample on miRCURY LNA miRNome Human Panels I and II (730 miRNAs in total). Data from 424 miRNAs with Cp values less than 37 are included. Normalized expression is shown as fold changes in tumor compared to normal. Out of 424 miRNAs expressed, 144 were more than 2-fold down-regulated in the tumor (blue dots) and 26 were more than 2-fold up-regulated in the tumor (red dots).