Isolation of circulating and EV-associated DNA from plasma and serum | EXO-DNAc-PS-20

miRNAs are small noncoding RNAs (approximately 19 to 22 nucleotides) that regulate protein expression and exert
physiological significance in several key cellular processes, such as cell differentiation, proliferation, and apoptosis. 1,2 Circulating miRNAs, which can be readily detected in biofluids, such as serum, plasma, or whole blood, are promising liquid biopsy biomarkers for noninvasive detection of various diseases, including cancer.

In addition, aberrations affecting miRNAs have been shown to significantly affect cancer genesis and progression. Because of their stability in serum/plasma, substantial attention and tremendous efforts have been dedicated to identify miRNA biomarkers for early detection, prognosis, or therapeutic purposes. However, changes in the miRNA expression level might be subtle during the onset of disease, thus making diagnosis challenging.  An ideal liquid biopsy biomarker should have a high signal/noise ratio between cancer and control samples, which can be readily detectable in clinical settings.

• Extracellular vesicles (EVs) play an important role in cellular communication and promote tumor development.  miRNA expression in EVs is frequently dysregulated and is potentially useful for early diagnosis of cancer or other diseases.
• Because EVs are key sources of circulating miRNAs in blood serum, it was hypothesized that isolation of EVs would enrich for miRNA biomarkers, leading to enhanced signal/noise ratios and thus improved diagnostic performance
• Five different isolation methods were evaluated, and polymer-based precipitation (PBP) approach was selected to isolate EVs from 15 GC and 15 matched healthy control serum samples. As a pilot study, a panel of 133 GC-related miRNAs was measured in both the total serum and EV fractions.
• Of these miRNAs, 11 were significantly different between cancer and control. In a separate validation set using 20 independent pairs of cancer and control serum, 8 of the 11 candidates were found to enhance the diagnostic
  potential of miRNA in EV fractions compared with total serum.
• Overall, it was demonstrated that the enrichment of miRNAs in EVs can significantly enhance the sensitivity of miRNA biomarkers in detecting GC, suggesting the potential use of EV-miRNAs in the diagnosis of GC.

Plasma/Serum Samples

Pooled normal human serum was purchased from InnovativeResearch (Novi, MI). Gastric cancer and healthy control serum samples were purchased from BioIVT (Westbury, NY).

EV Isolation from Serum

Serum was subjected to preclearing steps before EV isolation by centrifugation at 2000 g for 20 minutes, followed
by 10,000 g for 30 minutes. EVs were then isolated from 200 mL precleared serum.Ultracentrifugation Pre cleared serum (200 mL) was centrifuged at 100,000 g for 70 minutes at 4C using Optima MAX-XP Ultracentrifuge (Beckman Coulter, Brea, CA). Supernatant was aspirated, and the pellet was washed and recentrifuged at 100,000 g for 70 minutes at 4C. EV-containing pellet was resuspended in 200 mL phosphate-buffered saline (PBS) for subsequent RNA extraction. For protein analysis, the pellet was dissolved in 30 mL 5% SDS.

Polymer-Based Precipitation

EVs were isolated from 200 mL precleared serum using four commercial polymer-based precipitation reagents: Total Exosome Isolation from serum [Invitrogen (Invt), Carlsbad, CA], ExoQuick Exosome Precipitation Solution [SystemBiosciences (SBI), Palo Alto, CA], miRCURY Exosome Isolation KiteSerum and Plasma [Exiqon (Exi), Vedbaek,Denmark], and EXO-prep [HansaBioMed (Han), Tallinn, Estonia] were performed according to manufacturer’s protocol. EV pellets were resuspended in 200 mL PBS for subsequent RNA extraction. Pellet was dissolved in 30 mL 5% SDS for protein analysis.

Column AffinityeBased Purification

EVs were isolated from 200 mL precleared serum using exoRNeasy Serum/Plasma Midi Kit (Qiagen, Hilden, Germany) following manufacturer’s protocol. Briefly, serum was mixed with binding buffer and loaded onto the membrane column for washing. EVs were then directly lysed by addition of QIAzol (Qiagen), and RNA was eluted in 30 mL nuclease-free water. Peptide AffinityeBased Purification EVs were isolated using ME Kit (New England Peptide,Gardner, MA). Precleared serum (200 mL) was incubated with 20 mL Vn96 peptide stock overnight at 4C with end-to-end rotation. The mixture was centrifuged at 17,000 g at room temperature for 15 minutes. The supernatant was then removed, and EV-containing pellet was washedtwice with 500 mL PBS at 17,000 g for 10 minutes. EV-containing pellet was resuspended in 200 mL PBS for subsequent RNA extraction.

Immuno AffinityeBased Purification

EVs were isolated using ExoCap Composite Kit for Serum Plasma (JSR Life Sciences, Tokyo, Japan). Capture beads
(100 mL) were mixed with 1 mL treatment buffer and incubated with 200 mL precleared serum for overnight at
4C with end-to-end rotation. The supernatant was removed by placing the tube on a magnetic tube stand for 1 minute. Beads were washed twice with 500 mL washing/dilution buffer. Washed beads were resuspended in 200 mL PBS and proceeded to RNA extraction immediately.

RNA Isolation from Serum or EV Preparations

Total RNA from 200 mL EV preparations or 200 mL neat serum was extracted using miRNeasy serum/plasma miRNA isolation kit (Qiagen) according to the manufacturer’s protocol. For normalization of technical variations during RNA isolation, 1 mL of QIAzol lysis buffer was spiked with a set of three proprietary synthetic miRNAs (MiRXES,Singapore) before being added to the samples.

Subsequently, 200 mL chloroform was added to the mixture, thoroughly mixed, and centrifuged at 18,000 g for 15 minutes to allow phase separation. The resulting aqueousphase from each sample was transferred to QiaCube (Qiagen) for automated RNA binding, washing, and elution.RNA was eluted with 30 mL nuclease-free water.