METHODS

Blood from self-declared healthy donors was collected into EDTA, ACD-A, citrate, Nucleic Acid BCT, and Protein Plus BCT and immediately mixed end-over-end 10 times. Following draw or after 1, 3, or 5 days of ambient temperature storage, tubes were remixed end-over-end 10 times, then samples were processed to plasma using a double-spin protocol (1800 xg for 15 min, 2800 xg for 15 min).

For the RNA extraction kit comparison study, plasma was pooled from all donors (n=5), aliquoted, and frozen at -80 ˚C until use. RNA extractions were executed per the manufacturer’s protocols, where plasma input volume per RNA extraction kit was as follows:

• 500 μL – Plasma/Serum Circulating and Exosomal RNA Purification Kit (Slurry Format; Norgen, 51000)
• 200 or 500 μL – Maxwell^® RSC miRNA Plasma and Serum Kit (Promega Corporation, AS1680)
• 200 μL – QIAGEN miRNeasy Serum/Plasma Kit (QIAGEN, 217184)

For RNA spike-in experiments, 0.1 μL UniSP2/4/5 (QIAGEN, 339290) per 100 μL plasma was added to the lysis reagent.

For the miRNA stability study, RNA was extracted from 200 μL of individual plasma samples using the Promega kit.

For each tube and timepoint, extraction was performed in triplicate for the RNA extraction kit study and in singlicate for the miRNA stability study, and PCR was run in duplicate. All PCR assay outcomes were plotted without averaging. miRNAs were quantified by qRT-PCR using the miRCURY^® LNA^® RT and SYBR™ Green PCR Kits and miRCURY LNA miRNA PCR Assays (QIAGEN). The plots display Ct (Cycle threshold) values adjusted to equalize the differences in the plasma input and elution volumes of RNA extraction.

Maxwell Systems and the Maxwell RSC miRNA Plasma and Serum Kit are for Research Use Only.
Not for use in diagnostic procedures.

Figure 1. Blood from self-declared healthy donors was collected into EDTA, ACD-A, citrate, Nucleic Acid BCT, or Protein Plus BCT. At draw or after 1, 3, or 5 days at ambient temperature, plasma was isolated, then miRNA or mRNA was extracted and analyzed.