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[edit]

  • [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.

Part 1: DNA extraction from plasma samples[edit]

  • There are three tubes of plasma for each sample. Dr. Zhang suggested to use 2 tubes for DNA extraction.
  • I measured volume of two 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.
  • 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[edit]

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[edit]

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)[edit]
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[edit]

  • 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[edit]

  • 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[edit]

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.