Table 1.
Overview of RT-qPCR approaches tested
| Approach | Plasma preparation | RT-reaction | Proceeding of cDNA | References |
|---|---|---|---|---|
| 1 | 5 µL plasma + 5 µL denaturing buffer Incubation at 75 °C for 5 min, cool on ice Ad 2 µL spikea Centrifugation at 10,000g for 10 min at 4 °C |
6 µL plasma preparation in a total volume of 15 µL | None | Zhao et al. [9] |
| 2 | 2.5 µL plasma + 2.5 µL denaturing buffer Ad 1 µL spikea |
5 µL plasma preparation in a total volume of 15 µL | Centrifugation at 10,000g for 10 min | Asaga et al. [7] Zheng et al. [12] |
| 3 | 2.5 µL plasma + 2.5 µL denaturing buffer Ad RNase inhibitor and RT-primer pool Incubation at 70 °C for 10 min |
Ad rest of RT-reaction mixture and 1 µL spikea (total 15 µL) | Centrifugtion at 10,000g for 10 min | Liu et al. [8] |
| 4 | 5 µL plasma + 5 µL denaturing buffer Ad RNase inhibitor and 2 µL spikea Incubation at 70 °C for 10 min, cool on ice Centrifugation at 10,000g for 10 min at 4 °C |
6 µL plasma preparation in a total volume of 15 µL | None |
Overview of different RT-qPCR approaches tested in order to perform direct plasma analysis of microRNA-levels. The source for inspiration to each test procedure is provided in the last column
aCel-miR-39 (2.75 × 10−12 M)