The plasma samples were spun at 10,000 X g at 4°C for 10 minutes to remove cellular debris prior to EV and RNA isolation. EVs were isolated from 200 μL of plasma using qEV size-exclusion column (Izon Science, Cambridge, MA). Eluate fractions (~500 μL/per fraction) were collected individually and the particle count was determined using the qNano (Izon Science, Cambridge, MA) for each fraction. The fractions containing EV particles were then pooled and concentrated to ~200 μL using Amicon 10K centrifugation filters (EMD Millipore, Billerica, MA). RNA was isolated from either 200 μL of concentrated EVs, or 200 μL of whole plasma using the miRNeasy kit (QIAGEN, Germantown MD) according to the manufacturer’s instructions. To characterize the miRNA spectrum in EVs and whole plasma, a modified small-RNAseq (sRNAseq) library construction protocol was used9 (link).
Amicon 10k centrifugation filters
Manufactured by Merck Group
Sourced in Morocco
The Amicon 10K centrifugation filters are a type of lab equipment used for separating and concentrating macromolecules, such as proteins, from a solution. The filters have a molecular weight cut-off of 10,000 Daltons, which means they can retain molecules larger than 10,000 Daltons while allowing smaller molecules to pass through. The filters are designed to be used in a centrifuge, where the applied centrifugal force drives the solution through the filter membrane, leaving the desired macromolecules behind.
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Lab products found in correlation
2 protocols using amicon 10k centrifugation filters
1
Plasma-derived Extracellular Vesicle RNA Analysis
2
Urine Extracellular Vesicle Isolation
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EVs were isolated from 250 μl of urine using qEV size-exclusion column (Izon Science, Cambridge, MA) with de-gassed 1X phosphate-buffered saline. Eluate fractions (∼500 μl) containing microvesicles (fractions 7–10) were collected individually. The subsequent fractions depleted of microvesicles (11–35) were collected into a single 15-ml tube. The vesicle fractions were pooled and concentrated to ∼100 μl using Amicon 10K centrifugation filters (EMD Millipore, Billerica, MA) spun in a swing-bucket rotor at 4000g at 4°C for 20 minutes. To confirm the purification of EVs from samples, the qEV-purified EVs were examined with transmission electron microscopy at the Fred Hutchinson Cancer Center, as previously described.25
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