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Table 1 Overview of RT-qPCR approaches tested

From: Quantification of microRNA in plasma using probe based TaqMan assays: is microRNA purification required?

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  
  1. 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
  2. aCel-miR-39 (2.75 × 10−12 M)