Large-volume circulating nucleic acid extraction from serum, plasma and other cell-free liquid samples
Cat. No. R431601 / R431602 / R431603
For blood-derived input, centrifuge at 1,900 × g for 10 min at 4°C and transfer the plasma or serum to a new tube. For prepared serum, plasma or other cell-free liquid samples, begin with the clarified sample fraction.
Use EDTA or a cell-free nucleic acid preservation tube where applicable. Avoid repeated freeze–thaw cycles because circulating nucleic acids are easily degraded.
Centrifuge the plasma or serum at 4,000–5,000 × g for 15 min at 4°C to further remove cell debris and other particulate impurities. Transfer 1–5 ml supernatant to a new centrifuge tube.
This clarification step is important for reducing column clogging and background cellular nucleic acids.
Add 300 µl Buffer CFL per 1 ml plasma or serum. Vortex to mix thoroughly and incubate at room temperature for 10–15 min.
The displayed timeline uses the midpoint of the 10–15 min protocol range plus routine handling time.
For DNA-oriented recovery, add 30 µl Proteinase K per 1 ml plasma or serum, mix by inversion and incubate at 37°C for 30 min.
This optional step is described in Protocol 1 as a way to improve DNA production; it is not included in the standard cumulative timeline.
Add 100 µl Buffer CFP per 1 ml sample, vortex at high speed for more than 20 s and place on ice for 3 min.
Fully disperse the precipitate; nucleic acids can be trapped in sediment if this step is not mixed thoroughly.
Centrifuge at 13,000 × g for 5 min, then transfer the supernatant to a new centrifuge tube.
The manual also allows 4,000–5,000 × g for 15 min; the standard timeline uses the shorter high-speed route.
Add an equal volume of pre-cooled isopropanol containing 2% glacial acetic acid to the supernatant and vortex for 15 s.
Pre-cooled alcohol and acidified binding conditions support capture of low-abundance circulating nucleic acids.
Insert a HiPure Viral Midi Column into a 15 ml collection tube. Load less than 4 ml mixture per round and centrifuge at 4,000–5,000 × g for 3 min. Repeat loading until all mixture has passed through the column.
Repeated loading is included in the cumulative time and is most relevant for larger 1–5 ml inputs. Smaller sample inputs may require fewer loading cycles.
Add 3 ml Buffer MGW1 to the Viral Midi Column and centrifuge at 4,000–5,000 × g for 3 min. Discard filtrate and reinsert the column.
Buffer MGW1 must be prepared with isopropanol according to the bottle label before use.
Add 3 ml Buffer RW2 to the Viral Midi Column and centrifuge at 4,000–5,000 × g for 15 min.
This longer spin helps remove residual wash solution before transfer to the RNA Micro Column stage.
Transfer the column to a new 15 ml tube. Add 400 µl RNase Free Water to the membrane center, place for 5 min and centrifuge at 4,000–5,000 × g for 3 min.
This eluate is then reconditioned for the downstream RNA Micro Column concentration step.
Add 200 µl Buffer CFL and 0.9 ml absolute ethanol to the eluate, then vortex for 10 s.
This creates the binding condition for the smaller RNA Micro Column concentration stage.
Insert a HiPure RNA Micro Column into a 2 ml collection tube. Load up to 750 µl mixture and centrifuge at 12,000 × g for 1 min. Discard filtrate and repeat with the remaining mixture.
The two-load format is included in the cumulative time.
Add 600 µl Buffer RW2 and centrifuge at 12,000 × g for 30–60 s. Discard filtrate and repeat once.
The displayed timeline uses the midpoint of the 30–60 s spin range plus handling time. Buffer RW2 must be diluted with ethanol before use.
Centrifuge the RNA Micro Column at 12,000 × g for 3 min. Open the column lid and place at room temperature for 10 min to dry the membrane.
This step is important for reducing residual ethanol carryover into the final eluate.
Transfer the column to a 1.5 ml tube. Add 20–50 µl RNase Free Water to the membrane center, place for 2 min and centrifuge at 12,000 × g for 1 min.
Use a volume appropriate for downstream concentration requirements.
Discard the column and store the recovered nucleic acid at −20°C or −80°C.
Avoid repeated freeze–thaw cycles after elution.
For blood-derived input, centrifuge at 1,900 × g for 10 min at 4°C and transfer the plasma or serum to a new tube. For prepared serum, plasma or other cell-free liquid samples, begin with the clarified sample fraction.
Use EDTA or a cell-free nucleic acid preservation tube where applicable. Avoid repeated freeze–thaw cycles for circulating RNA work.
Centrifuge the plasma or serum at 4,000–5,000 × g for 15 min at 4°C to further remove cell debris and transfer 1–5 ml supernatant to a new tube.
Clarification helps reduce cellular debris before the large-volume column step.
Add 300 µl Buffer CFL per 1 ml plasma or serum. Vortex to mix thoroughly and incubate at room temperature for 10–15 min.
The displayed timeline uses the midpoint of the 10–15 min protocol range plus routine handling time.
Add 100 µl Buffer CFP per 1 ml sample, vortex at high speed for more than 20 s and place on ice for 3 min.
Fully disperse the precipitate to avoid losing nucleic acids with sediment.
Centrifuge at 13,000 × g for 5 min and transfer the supernatant to a new centrifuge tube.
The manual also allows 4,000–5,000 × g for 15 min; the standard timeline uses the shorter high-speed route.
Add an equal volume of pre-cooled isopropanol containing 2% glacial acetic acid to the supernatant and vortex for 15 s.
Proceed promptly after mixing to the Viral Midi Column binding step.
Insert a HiPure Viral Midi Column into a 15 ml collection tube. Load less than 4 ml mixture per round and centrifuge at 4,000–5,000 × g for 3 min. Repeat until all mixture has passed through the column.
Repeated loading is included in the cumulative time and depends on sample input volume.
Add 3 ml Buffer MGW1 and centrifuge at 4,000–5,000 × g for 3 min. Discard filtrate and reinsert the column.
Buffer MGW1 must be prepared with isopropanol before use.
Add 3 ml Buffer RW2 and centrifuge at 4,000–5,000 × g for 10 min.
Protocol 2 uses a 10 min RW2 spin at the Viral Midi Column stage.
Transfer the column to a new 15 ml tube. Add 600 µl Buffer CFL to the membrane center, place for 5 min and centrifuge at 4,000–5,000 × g for 3 min.
This eluate is directly adjusted with ethanol for RNA Micro Column binding.
Add 0.9 ml absolute ethanol to the eluate and vortex for 10 s.
Do not omit ethanol before loading onto the RNA Micro Column.
Insert a HiPure RNA Micro Column into a 2 ml collection tube. Load up to 750 µl mixture and centrifuge at 12,000 × g for 1 min. Discard filtrate and repeat with the remaining mixture.
The two-load format is included in the cumulative time.
Add 600 µl Buffer RW2 and centrifuge at 12,000 × g for 30–60 s. Discard filtrate and repeat once.
The displayed timeline uses the midpoint of the 30–60 s range plus handling time.
Centrifuge at 12,000 × g for 3 min. Open the column lid and place at room temperature for 10 min to dry the membrane.
Drying helps reduce ethanol carryover into RNA eluate.
Transfer the column to a 1.5 ml tube. Add 20–50 µl RNase Free Water to the membrane center, place for 2 min and centrifuge at 12,000 × g for 1 min.
Use RNase-free handling throughout this final stage.
Discard the column and store RNA at −20°C or −80°C.
Avoid repeated freeze–thaw cycles after elution.
1. Workflow structure. This workflow separates plasma / serum clarification, large-volume circulating nucleic acid capture on the HiPure Viral Midi Column, and final concentration on the HiPure RNA Micro Column. It is intended as a practical companion to the product manual rather than a replacement for the official protocol.
2. Time interpretation. Protocol times stated in the product manual are retained where applicable. Steps without explicit timing are estimated for an experienced operator, including pipetting, tube transfer, repeated column loading, filtrate removal and column repositioning. For short protocol ranges, the timeline uses the midpoint. For long or optional protocol ranges, the displayed standard timeline uses the shortest reasonable path, while the note and total-time range indicate where extended handling may apply. Cumulative time runs continuously from the first clarification step to final elution.
3. Workflow characteristics. R4316 is a two-column workflow for low-abundance circulating nucleic acids. The first Viral Midi Column handles large-volume serum or plasma lysate after precipitation and alcohol adjustment. The intermediate eluate is then reconditioned and concentrated through the RNA Micro Column, allowing recovery into a small final RNase Free Water volume.
4. Practical considerations. The key handling points are complete plasma / serum clarification, thorough mixing after Buffer CFP addition, correct preparation of Buffer MGW1 and Buffer RW2, and careful drying of the RNA Micro Column before final elution. Proteinase K treatment in Protocol 1 is optional and can improve DNA production, but it adds about 30 minutes and is not included in the standard displayed timeline. If the lower-speed centrifugation option is used instead of 13,000 × g clarification, or if a full 5 ml input requires additional loading rounds, total processing time may move toward the upper end of the range.