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Centrifuge at 12,000× g for 3 min at room temperature. This should result in a clear and colorless
supernatant. Transfer the supernatant to a new microcentrifuge tube, add an equal volume of 100%
isopropanol, and vortex for 5 s. Transfer the full volume (approximately 500 µL) into a miRNeasy UCP
MinElute column nested inside a clean, 1.5 mL microcentrifuge tube and spin at 8,000× g for 15 s. Discard
the flow-through. Note: To perform the wash steps, add subsequent volumes of reagents directly into the center
of the column. Add 700 µL of buffer RWT, centrifuge for 15 s at 8,000× g and discard the flow-through.
Next, add 500 µL of buffer RPE, centrifuge for 15 s, and discard flow-through. Add an additional 500 µL of
80% ethanol and centrifuge for 2 min. Discard the flow-through. Transfer the miRNeasy UCP MinElute
spin column to a new collection tube and leave the lid open. Centrifuge at full speed for 5 min; discard any
remaining flow-through and the collection tube. Place the miRNeasy UCP MinElute spin column into a
new 1.5 mL collection tube and add 20 µL of water directly to the center of the spin column membrane.
Incubate for 10 min at room temperature. Finally, centrifuge for 1 min at full speed to elute the isolated
miRNA. Note: 18 µL of total miRNA is normally recovered following elution. Proceed to small RNA
quantification and concentration normalization immediately.

4. Small RNA quantification and concentration normalization [Figure 2C]:

Using 1 µL of the eluted miRNA, quantify concentration in ng/µL using the Qubit miRNA Assay and
following the manufacturer’s instructions. After the concentration is measured, dilute the miRNA with
RNase-free water such that the miRNA concentration will be 122.55 ng/µL.

Figure 3 represents expected miRNA concentrations following quantification using the Qubit miRNA assay.
Median values obtained from 47 different healthy human subjects were 1,062 ± 202.01 ng/µL. The standard
deviation was 1,384.90 ng/µL. The 25th and 75th interquartile ranges were 760 and 1,458 ng/µL, respectively,
with a coefficient of variation of 13.54. Note: If measured values fall outside the range reported in Figure 3,
results likely indicate degradation or contamination. Compromised samples with concentrations outside these
ranges must be excluded from subsequent analysis.

Once you have completed the above steps, roughly 18,054 ng of total miRNA remains for analysis. This
allows for quantification of approximately 9 miRNA targets per 200 µL of plasma. Proceed to spike-in
addition and miRNA-specific cDNA generation immediately.

5. Spike-in addition and miRNA-specific cDNA generation [Figure 2D]:

Once total miRNA has been corrected to a concentration of 122.55 ng/µL proceed to cDNA generation. For
cDNA generation, a miR-39 counterpart is prepared separately for each miRNA target under investigation.
Target-specific and miR-39-specific cDNA generation will be performed separately to avoid cross-reactivity.
Supplementary Figure 1 is an example of results when cDNA generation is performed in a single tube. The
skewed negative population resulting from the cross-reactivity impedes the ability to accurately set a
threshold and acquire reproducible data.

miRNA target specific cDNAs will be generated using the TaqMan miRNA Reverse Transcription Kit
(Catalog number: 4366597) with TaqMan miRNA specific assays (Catalog number: 4427975). We follow all
instructions from the manufacturers, which can be found in the user manuals. The following is a summary
that includes precise numerical values for optimized reaction volumes and conditions.