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 another cell-free liquid sample, begin with the clarified sample fraction.
Use EDTA or a cell-free nucleic acid preservation tube where applicable. Avoid repeated freeze–thaw cycles.
Centrifuge 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.
Do not disturb the residual pellet. Complete clarification reduces column clogging and background cellular nucleic acids.
Add 300 µl Buffer CFL per 1 ml plasma or serum, vortex until mixed and incubate at room temperature for 10–15 min.
The displayed time uses a near-midpoint incubation plus routine addition and mixing time.
Add 30 µl Proteinase K per 1 ml plasma or serum, mix by inversion and incubate at 37°C for 30 min.
The manual identifies this option as a way to improve DNA yield. 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.
A large precipitate forms at this stage. Disperse it thoroughly so nucleic acids are not trapped and lost with the sediment.
Centrifuge at 13,000 × g for 5 min at room temperature and transfer the supernatant to a new centrifuge tube.
The manual also permits 4,000–5,000 × g for 15 min. The standard timeline uses the shorter high-speed route.
Add an equal volume of ice-cooled isopropanol containing 2% glacial acetic acid to the supernatant and vortex for 15 s.
Prepare the acidified isopropanol in advance by mixing 9.8 ml isopropanol with 0.2 ml glacial acetic acid and cooling it on ice.
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 at room temperature. Discard the filtrate and repeat until the entire mixture has passed through the column.
The number of loading rounds depends on the initial 1–5 ml sample volume. The displayed timeline represents a larger-volume preparation.
Add 3 ml Buffer MGW1 and centrifuge at 4,000–5,000 × g for 3 min. Discard the filtrate and return the column to the collection tube.
Buffer MGW1 must be prepared with isopropanol according to the bottle label.
Add 3 ml Buffer RW2 and centrifuge at 4,000–5,000 × g for 15 min. Discard the filtrate and transfer the column to a new 15 ml tube.
Buffer RW2 must be diluted with ethanol before use.
Add 600 µl RNase Free Water to the membrane center, let stand for 5 min and centrifuge at 4,000–5,000 × g for 3 min. Discard the Viral Midi Column and retain the eluate.
The eluate is reconditioned for concentration on the RNA Micro Column.
Add 250 µl Buffer CFL and 0.75 ml isopropanol to the eluate and vortex for 10 s.
Use isopropanol at this stage; the revised Protocol 1 no longer specifies absolute ethanol for micro-column re-binding.
Insert a HiPure RNA Micro Column into a 2 ml collection tube. Load no more than 750 µl mixture and centrifuge at 12,000 × g for 1 min. Discard the filtrate and repeat until the entire mixture has passed through the column.
The nominal mixture may require more than two loading cycles. Never exceed 750 µl in a single load.
Add 500 µl Buffer RW2 and centrifuge at 12,000 × g for 30–60 s. Discard the filtrate and repeat once.
The displayed time uses a near-midpoint spin time plus loading, filtrate disposal and column repositioning.
Centrifuge at 12,000 × g for 3 min, then open the column lid and leave the column at room temperature for 10 min.
Complete drying reduces residual ethanol carryover into the final eluate.
Transfer the column to a new 1.5 ml tube. Add 15–50 µl RNase Free Water to the membrane center, let stand for 2 min and centrifuge at 12,000 × g for 1 min.
Select the elution volume according to the concentration required for downstream analysis.
Discard the column and store the recovered cfDNA/RNA 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 another cell-free liquid sample, begin with the clarified sample fraction.
Use EDTA or a cell-free nucleic acid preservation tube where applicable. Avoid repeated freeze–thaw cycles.
Centrifuge at 4,000–5,000 × g for 15 min at 4°C to further remove cell debris. Transfer 1–5 ml supernatant to a new centrifuge tube.
Do not disturb the residual pellet when transferring the clarified fraction.
Add 300 µl Buffer CFL per 1 ml plasma or serum, vortex until mixed and incubate at room temperature for 10–15 min.
The displayed time uses a near-midpoint incubation plus routine handling.
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.
Disperse the precipitate thoroughly to avoid trapping nucleic acids in the sediment.
Centrifuge at 13,000 × g for 5 min and transfer the supernatant to a new centrifuge tube.
The manual also permits 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 to the Viral Midi Column binding step after mixing.
Load less than 4 ml mixture per round and centrifuge at 4,000–5,000 × g for 3 min. Discard the filtrate and repeat until the entire mixture has passed through the column.
The number of loading rounds depends on the initial sample volume.
Add 3 ml Buffer MGW1 and centrifuge at 4,000–5,000 × g for 3 min. Discard the filtrate and return the column to the collection tube.
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. Discard the filtrate and transfer the column to a new 15 ml tube.
Protocol 2 uses a 10 min RW2 spin at this stage.
Add 600 µl Buffer CFL to the membrane center, let stand for 5 min and centrifuge at 4,000–5,000 × g for 3 min. Discard the column and retain the eluate.
Buffer CFL in the eluate supports the subsequent RNA Micro Column route.
Add 0.9 ml absolute ethanol and vortex for 10 s.
Do not omit ethanol before loading the RNA Micro Column.
Load no more than 750 µl mixture and centrifuge at 12,000 × g for 1 min. Discard the filtrate and repeat with the remaining mixture.
The revised manual specifies two loading steps for this 1.5 ml mixture.
Prepare 80 µl DNase I mixture using 70 µl DNase Buffer and 10 µl DNase I (10 units/µl). Apply the mixture to the membrane center and incubate at room temperature for 15 min.
DNase I Set B (Cat. No. C12138) is not supplied. Although the procedure labels this step optional, it is included in this timeline because DNA removal defines the DNA-depleted cfRNA route.
Add 600 µl Buffer MGW1, let stand at room temperature for 1 min and centrifuge at 12,000 × g for 1 min.
Retain the filtrate for the next step rather than discarding it.
Transfer the column to a new 2 ml collection tube, return the retained filtrate to the column and centrifuge at 12,000 × g for 1 min. Discard the filtrate and return the column to the collection tube.
This second pass is part of the revised post-DNase purification route.
Add 600 µl Buffer RW2 and centrifuge at 12,000 × g for 30–60 s. Discard the filtrate and repeat once.
Buffer RW2 must be diluted with ethanol before use.
Centrifuge at 12,000 × g for 3 min, then open the column lid and leave the column at room temperature for 10 min.
Remove residual wash solution without touching the membrane or contaminating the column.
Transfer the column to a 1.5 ml tube. Add 20–50 µl RNase Free Water to the membrane center, let stand for 2 min and centrifuge at 12,000 × g for 1 min.
Use RNase-free handling during elution and transfer.
Discard the column and store the recovered cfRNA at −20°C or −80°C.
If DNase treatment is omitted, subtract approximately 24 min from the displayed route; the recovered RNA fraction has not then been actively depleted of DNA.
1. Workflow structure. This workflow separates plasma / serum clarification, metal salt-mediated protein precipitation, large-volume capture on the HiPure Viral Midi Column, and final concentration on the HiPure RNA Micro Column. Protocol 1 co-purifies circulating DNA and RNA, including miRNA. Protocol 2 uses the revised on-column DNase, MGW1 and filtrate-return sequence to produce a DNA-depleted circulating RNA fraction. This note is 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 reagent addition, vortexing, tube transfer, repeated column loading, centrifuge loading and unloading, filtrate removal and column repositioning. Short protocol ranges use a midpoint or near-midpoint value. The standard timeline uses the 13,000 × g sediment-removal route and a larger-volume loading estimate. Protocol 2 includes the 15 min DNase incubation and its associated wash and filtrate-return steps because they define the DNA-depleted cfRNA route. Cumulative time runs continuously from the first clarification step to final storage.
3. Workflow characteristics. R4316 uses a two-stage spin-column procedure for low-abundance cell-free nucleic acids from 1–5 ml samples. The Viral Midi Column captures nucleic acids from the precipitated, acidified-isopropanol-adjusted sample. Protocol 1 elutes with RNase Free Water and reconditions the eluate with Buffer CFL and isopropanol before micro-column concentration. Protocol 2 elutes with Buffer CFL, adjusts with ethanol and performs DNase treatment on the RNA Micro Column before final washing and elution.
4. Practical considerations. Thoroughly clarify plasma or serum, fully disperse the precipitate after Buffer CFP addition and avoid transferring sediment. Prepare acidified isopropanol, Buffer MGW1 and Buffer RW2 correctly before starting. Never exceed the stated loading volume per column cycle. In Protocol 2, retain the MGW1 filtrate after the post-DNase wash and pass it through the RNA Micro Column again as specified. Proteinase K in Protocol 1 remains optional and adds about 30 min. Omitting DNase from Protocol 2 shortens the route by about 24 min, but the resulting RNA fraction has not been actively depleted of DNA.