Page 145 - Read Online

Page 8 of 19 Maiocchi et al. Vessel Plus 2023;7:27 https://dx.doi.org/10.20517/2574-1209.2023.69

Figure 3. Expected range of microRNA concentration following isolation. microRNA is isolated from plasma with the Qiagen miRNeasy
Serum/Plasma Advanced Kit, and the concentration of microRNA is quantified by the Qubit microRNA assay. Results represent 47
independent biological samples where the dotted line is the mean, and the solid line is the median, and the bar represents the
interquartile range.

Prepare a 5X stock concentration of all TaqMan miRNA specific reverse transcriptase (RT) primers. Vortex
and spin-down all reaction components on a tabletop centrifuge. For each miRNA target: separately prepare
a target-specific cDNA and miR-39 specific cDNA reaction using 1 µg of total miRNA for each reaction.
Each master mix is created by combining the following components per reaction: 0.15 µL of dNTP Mix,
0.19 µL of RNase Inhibitor, 1.5 µL of 10X RT Buffer, 1 µL of Multiscribe Reverse Transcriptase, and 3 µL of
the 5X target-specific RT primer, or the 5X miR-39 specific RT primer.

Following master mix preparation, pipette 5.84 µL of the designated master mix and add 8.16 µL of the
122.55 ng/µL miRNA dilution into each corresponding tube for a total volume of 14 µL.

Next, vortex the selected miR-39 spike-in for 30 s and rest it on ice for 60 s. Repeat this process three times.
Then, add 1 µL of the miR-39 spike-in to the respective reactions for a total volume of 15 µL. Please note: the
only difference between miRNA target specific and miR-39 specific reactions is the RT probe.