Noi/NOTES/2014-6-9

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DNA extraction of 10 plasma samples and 5 matched pair solid tumor tissue samples from Cancer Center

  • [Link to calendar]
  • Part 1: I extract 10 plasma DNA samples from cancer patients received on 2014-06-05
  • Part 2: I extract 5 matched pair solid tumor tissue samples from cancer patients received on 2014-05-27
  • I assigned new sample codes to be more convenient for sample handling. --> In the future, I may only use the last 3 digit of project ID, so I don't need to rerun sample code and prevent confusion.
  • I measured volume of the first tube serum during DNA extraction and adjust volume to 600ul with 1X PBS as ChargeSwitch kit required different volume of reagent for different sample volume.

Part 1: DNA extraction from plasma samples

  • There are three tubes of plasma for each sample. Dr. Zhang suggested to use 2 tubes for DNA extraction.
  • Note that the measurement could be +-5ul error since serum is viscous and I needed to adjust the pipette volume after aspiration to measure the volume
Project ID # Zhang lab ID Volume 2 tubes PBS to 600ul
UCSD-004-04 PC-P_1 560 40
UCSD-004-05 PC-P_2 475 125
UCSD-004-06 PC-P_3 475 125
UCSD-004-07 PC-P_4 330 270
UCSD-004-08 PC-P_5 475 125
UCSD-004-09 PC-P_6 480 120
UCSD-004-10 PC-P_7 480 120
UCSD-004-11 PC-P_8 450 150
UCSD-004-12 PC-P_9 600 0
UCSD-004-13 PC-P_10 440 160
  • I followed instruction of ChargeSwith gDNA 1 ml Serum kit as described in [[1]] for 600ul sample volume

Prep

  • Eluted 30ul with Elution Buffer
  • Measure concentration with Qubit dsDNA HS assay using 2ul of extracted DNA in 200ul reaction (100x dilution)

Qubit dsDNA HS assay result

Sample Conc. in the Qubit Unit uL used Dilution Sample conc. (ng/uL) Volume (ul) Yields (ng)
PC-P_1 4.73 ng/mL 2 100 0.473 30.00 14.19
PC-P_2 8.52 ng/mL 2 100 0.852 30.00 25.56
PC-P_3 13.6 ng/mL 2 100 1.360 30.00 40.80
PC-P_4 3.4 ng/mL 2 100 0.340 30.00 10.20
PC-P_5 8.13 ng/mL 2 100 0.813 30.00 24.39
PC-P_6 7.67 ng/mL 2 100 0.767 30.00 23.01
PC-P_7 6.44 ng/mL 2 100 0.644 30.00 19.32
PC-P_8 5.24 ng/mL 2 100 0.524 30.00 15.72
PC-P_9 2.38 ng/mL 2 100 0.238 30.00 7.14
PC-P_10 3.7 ng/mL 2 100 0.370 30.00 11.10

Normalize DNA yield to 1mL serum volume

Sample Yields (ng) Plasma volume (ul) Normalized yield to 1mL (ng)
PC-P_1 14.19 560 25.34
PC-P_2 25.56 475 53.81
PC-P_3 40.80 475 85.89
PC-P_4 10.20 330 30.91
PC-P_5 24.39 475 51.35
PC-P_6 23.01 480 47.94
PC-P_7 19.32 480 40.25
PC-P_8 15.72 450 34.93
PC-P_9 7.14 600 11.90
PC-P_10 11.10 440 25.23
Plasma color (in lysis buffer)
Row 1: PC-P_1-5
Row 2: PC-P_6-10
File:2014-06-14 PC-P 1-10color.jpg

Part 2: DNA extraction from solid tumor tissue samples

  • The size of tissue samples are varied. Sharmeela noted that they should be ~25mg.
  • I used DNeasy Blood and Tissue kit from QIAGEN and followed the instruction of the manufacturer.

Procedures

  • Followed protocol of DNeasy Blood and tissue kit: [[2]]. Page 28.
  • I added RNase A to remove RNA contamination
- Rui suggested that if the tissue is large, I should increase volume of ATL buffer ~3X.
- I added 180*3ul = 540ul of ATL buffer + 50ul of 10mg/mL RNase A (actually should right before Buffer AL) + 60ul of ProteinaseK
- Incubate at 56C for ~1h with shaking 900rpm/min and occasionally vigorously vortexing for every 10min.
I did not use homogenizer to homogenize tissue as it might be difficult to handle and it will generate a lot of bubbles.
- Anyway, Vortexing and shaking on thermo mixer is good enough to lyse the tissue.
- After the tissue is completely lysed, I split it into two tube (~330ul each) + added 330ul of Buffer AL
  • Eluted 1st time with 200ul AE Buffer
  • Eluted 2nd time with 50ul AE Buffer (total each column ~250ul
  • Pooled two tubes together --> total volume left ~480ul
  • Measure concentration with Qubit dsDNA HS assay using 2ul of extracted DNA in 200ul reaction (100x dilution). Samples PC-T_1 has very high concentration. I repeated measurement and reduced volume to 1ul.

Qubit dsDNA HS assay result

Sample Conc. In the Qubit Unit uL used Dilution Sample conc. (ng/ul) Volume (ul) Yield in 480ul (ug)
PC-T-1 413 ng/mL 1 200 82.7 480 39.70
PC-T-2 358 ng/mL 2 100 35.8 480 17.18
PC-T-4 428 ng/mL 2 100 42.8 480 20.54
PC-T-6 152 ng/mL 2 100 15.2 480 7.30
PC-T-7 163 ng/mL 2 100 16.3 480 7.82
  • I also measured with ND to determined DNA extraction quality and compare measurement by Qubit and ND
Sample ND conc. (ng/ul) 260/280 260/230
PC-T_1 89.30 1.91 2.25
PC-T_2 40.70 1.82 1.94
PC-T_4 44.20 1.89 2.12
PC-T_6 15.30 2.17 2.13
PC-T_7 16.70 1.81 1.92
  • The concentrations from both Qubit and ND are very close this meant that there is very low protein and salt contamination.