Noi/NOTES/2014-2-28

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MONOD test on human plasma/serum

  • Note from Dr. Zhang
" We will get some human plasma and serum from cancer center for the MONOD project. This would be different from all the BSPP experiments we did before, as we are dealing with circulating cell-free DNA from human patients.
One major challenge is to recover enough DNA for BSPP, as the yield of DNA extraction from plasma/serum is very low in general, and seems to be quite variable depending on the DNA extraction kits (we don’t want whole blood or buffy coats as they contain mostly white blood cells).
We will received a set of four tubes of plasma from one individual, and another four tubes of serum from another. Each tube has roughly 200ul of biofluid.
I’ve ordered four kits (QIAamp blood mini, Lifetech ChargeSwitch, Clontech Nucleospin, ZymoBead). Our first step is to determine the yields with these kits, and for the bead-based kits, to try direct bisulfite conversion of DNA on the beads without washing and elution.
Please keep track of the kits when they arrive, and write up an experimental protocol based on the manuals of these kits. The materials are precious so please confirm the plan with me before processing the samples. We will do picogreen quantification and Bioanalyzer analysis to determine the yield and size distribution (cell-free DNA are fragmented in general). We can move forward with BSPP if there is enough material.
We will received a set of four tubes of plasma from one individual, and another four tubes of serum from another. Each tube has roughly 200ul of biofluid ".
Kit Vendor Catalog # Protocol Note
QIAamp DNA Blood Mini Kit (50) QIAGEN 51104 QIAamp Blood Mini Kit Manual Column-based method, page 26-28
ChargeSwitch® gDNA Serum Kit, 0.2-1 mL Life Technologies CS11040 ChargeSwitch® gDNA Serum Kit Manual Bead-based method
NucleoSpin® Plasma XS Fisher Scientific/Marcherey Nagel NC9959409 NucleoSpin® Plasma XS kit manual Column-based method, Estimeted delivert date: 2014-03-14
ZymoBead™ Genomic DNA Kit Zymo Research D3004 ZymoBead™ Genomic DNA Kit manual Bead-based method

Experimental plan

  • I will confirm with Dr. Zhang about the volume of serum/plasma to use for each kit since each one uses different volume. Shall I used recommended volume of each kit and then compare the yield to normalized input?

Column-based methods

1. QIAamp DNA Blood Mini Kit

- Resuspend lyophilized QIAGEN Protease with 1.2 ml protease solvent.
- Use carrier DNA (e.g., poly dA, poly dT, poly dA:dT) when the sample is low-copy (i.e., when <10,000 copies are present)
Starting material:    200ul for blood, plasma, serum or buffy coat 

Procedures

Preparing lysate
- Pipet 20 µl QIAGEN Protease (or proteinase K) into the bottom of a 1.5 ml tube
- Add 200 µl sample to the tube. If the volume of sample less than 200ul, adjust volume with PBS
- Add 200 µl Buffer AL to the sample. Mix by pulse-vortexing for 15 s
- Incubate at 56°C for 10 min
Binding DNA
- Briefly spin down the tube before adding 200ul 100% EtOH. Mix again by pulse-vortexing for 15 s and briefly spin down
- Transfer spnt to QIAamp Mini spin column & centrifuge at 6,000xg for 1 min
Washing DNA
- Transfer QIAamp Mini spin column in a clean 2 ml collection tube
- Add 500 µl Buffer AW1 & centrifuge at 6,000xg for 1 min
- Transfer QIAamp Mini spin column in a clean 2 ml collection tube
- Add 500 µl Buffer AW2 & centrifuge at 20,000xg for 3min
Eluting DNA
- Transfer the QIAamp Mini spin column in a new 2 ml collection tube and centrifuge at 20,000xg for 1 min (full speed)
- Place the QIAamp Mini spin column in a clean 1.5 ml tube & add appropriate volume of Buffer AE or distilled water (manual recommends 200ul).
- Incubate at RT for 5 min & centrifuge at 6,000xg for 1 min


2. NucleoSpin® Plasma XS kit

Product specifications
Starting material:    Up to 240 μL EDTA plasma 
Elution volume:       5–30 μL
Average yield:        0.1–100 ng per mL plasma
DNA size range:       50-1000bp
DNA yield from human plasma: DNA amounts from less than 0.1 ng DNA per mL of plasma up to several 100 ng DNA per mL of plasma have been reported

Procedures

# To remove residual cells, centrifuge plasma/serum at >11,000xg for 3 min
Preparing lysate
- Add240ul of plasma/serum to 1.5mL tube.
- Add 20ul proteinase K to plasma/serum sample
- Mix and incubate at 37C for 10min
Binding DNA
- Add 1.5 volume (360ul) of buffer BB (keep sample:binding buffer ration 1:1.5 v/v)
- Invert the tube 3x and vortex for 3s. Spin down the tube briefly.
- Load 600ul of the mixture to a Nucleospin Plasma XS column
- Centrifuge at 2,000xg for 30s. Centrifuge at higher speed at 11,000xg for further 5s.
Washing DNA
- Transfer Nucleospin column to a new collecting tube
- Add 500ul of buffer WB onto the column. Centrifuge at 11,000xg for 30s.
- Place column into new collecting tube
- Add 250ul of buffer WB to the column. Centrifuge at 11,000xg for 3min
Eluting DNA
- Place column to 1.5mL tube
- Add 20ul Elution buffer to the column (range 5-30ul)
- Centrifuge at 11,000xg for 30s
- Incubate eluted DNA with open lid at 90C for 8min to remove residual ethanol

Bead-based methods

3. ChargeSwitch® gDNA 1 ml Serum kit

- Upon receipt, store proteinase K at 4C
Product Specifications
Starting Material:    0.2-1 ml of fresh or frozen human serum, with or without EDTA
Elution Volume:       50 μl
DNA Yield:            1-200 ng
DNA Size:             50 bp-1.5 kb

Procedures

Suggested reagent volumes for different sample volumes

Reagent\sample volume (S) 1mL 800ul 600ul 400ul 200ul
A volume: ChargeSwitch Lysis Buffer (L19) 700 ul 560 ul 420 ul 280 ul 140 ul
Proteinase K 30 ul 30 ul 30 ul 30 ul 30 ul
Rnase A (optional) 5 ul 5 ul 5 ul 5 ul 5 ul
B volume: ChargeSwitch Purification Buffer (N7) 250 ul 200 ul 150 ul 100 ul 50 ul
C volume: ChargeSwitch Magnetic beads 30 ul 24 ul 18 ul 12 ul 6 ul
D volume: ChargeSwitch Wash Buffer (W12) 1 mL 800 ul 600 ul 400 ul 200 ul
ChargeSwitch Elution Buffer (E5) 50 ul 50 ul 50 ul 50 ul 50 ul
  • Before starting, mix Lysis buffer with proteinase K according to table above & scale up volume for multiple samples
Preparing lysate
- Add S volume of sample to 1.5mL tube
- Add A volume + 30ul of Lysis mix (Lysis Buffer + proteinase K)
- Add 5ul of RNase A
- Pipette up & down 5x to mix
- Incubate at RT for 20min to lyse the sample. Alternatively incubate on a rotating mixer
Binding DNA
- Vortex the tube containing ChargeSwitch Magnetic beads
- Add B volume of ChargeSwitch Purification Buffer to the digested sample
- Add 30 ul of ChargeSwitch Magnetic beads and pipette up & down 5x to mix (use p1000 pipette, avoid generating bubbles)
- Incubate at RT for 2min to allow DNA bind to magnetic beads
- Place the sample tube in the MagnaRack for 3 min or until the bead forming a tight pellet. (I may use magnetic rack for 1.5mL tube available in the lab)
- Carefully remove the spnt without disturbing the bead pellet
Washing DNA
- Remove sample tube from magnetic rack
- Add D volume of ChargeSwitch Wash Buffer to the tube and pipette up & down 5x gently & avoid bubbles
- Place the sample tube in the MagnaRack for 2 min or until the bead forming a tight pellet
- Carefully remove the spnt without disturbing the bead pellet
- Repeat washing step
Eluting DNA
- Remove sample tube from magnetic rack (there should be no spnt in the tube)
- Add 50ul of ChargeSwitch Elution Buffer to the tube and pipette up & down gently 10x to resuspend magnetic beads
# Do not use H2O for elution
- Place the sample tube in the MagnaRack for 1 min or until the bead forming a tight pellet
- Carefully transfer eluted DNA to 1.5mL tube

4. ZymoBead™ Genomic DNA kit