Noi/NOTES/2014-6-27

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

Calendar

  • Part 1: I extract matched pair solid tumor tissue samples (colon & lung) from cancer patients received on 2014-06-27
  • Part 2: I extract plasma samples from cancer patients (colon & lung) received on 2014-06-26

Part 1: DNA extraction from solid tumor tissue samples

  • The size of tissue samples are varied. Sharmeela noted that they should be ~20mg.
  • 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: Page 28, but increase volume of reagents to 4x as the size of tissue is quite big.
  • I added RNase A to remove RNA contamination
- I added 170ul* 4 = 680ul of ATL buffer + 80ul of ProteinaseK
- Incubate at 56C for ~2-3h 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. This time took longer to lyse the colon and lung tissues than pancreas tissue. Possibly the tissue compositions are differents.
- After the tissue is completely lysed, I add 40ul of 10mg/mL RNase A to each tub and incubate at RT for 2min (I'm supposed to add 80ul, but I do not have enough RNaseA)
- I split it into two 2mL tube (~400ul each) + added 4000ul of Buffer AL. Mix vigorously.
- Add 800ul of 100% EtOH. Mix Vigorously.
- Load cell lysis through column ~800ul (reload more 800ul --> total 1600ul of cell lysis/ column)
- Wash with 500ul AW1 Buffer
- Wash with 500ul of AW2 Buffer
- Eluted 1st time with 100ul AE Buffer
- Eluted 2nd time with 100ul AE Buffer (total each column ~200ul
- Pooled two tubes together --> total volume left ~400ul
  • 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) Yield in 400ul (ug)
6-T_1 434 ng/mL 2 100 43.40 400.00 17.36
6-T_2 316 ng/mL 2 100 31.60 400.00 12.64
6-T_3 315 ng/mL 2 100 31.50 400.00 12.60
6-T_4 417 ng/mL 2 100 41.70 400.00 16.68
6-T_5 305 ng/mL 2 100 30.50 400.00 12.20
7-T_1 307 ng/mL 2 100 30.70 400.00 12.28
7-T_2 446 ng/mL 2 100 44.60 400.00 17.84
7-T_3 359 ng/mL 2 100 35.90 400.00 14.36
7-T_4 338 ng/mL 2 100 33.80 400.00 13.52
7-T_5 204 ng/mL 2 100 20.40 400.00 8.16
  • I also measured with ND to determined DNA extraction quality and compare measurement by Qubit and ND
Sample ID ND conc. (ng/ul) 260/280 260/230
6-T-1 57.51 2 1.67
6-T-2 84.83 1.99 1.54
6-T-3 42.86 1.84 1.04
6-T-4 55.42 1.69 0.96
6-T-5 36.96 1.93 1.26
7-T-1 39.44 1.51 0.89
7-T-2 66.63 1.78 1.36
7-T-3 62.39 1.83 1.37
7-T-4 49.6 1.91 1.53
7-T-5 28.74 1.68 1.07
  • The concentrations from both Qubit and ND are not too much different. However the ratio of 260/230 is quite low in most samples. I am not sure if this because I reloaded column with double amount of lysed cells.
  • The total of gDNA extracted from tumor tissue is not very high compare to the size of tissue. I think the majority of the tissue in more fibers and the components of connective tissue.

2014-06-28

Part 2: DNA extraction from plasma samples

  • 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
  • I followed instruction of ChargeSwith gDNA 1 ml Serum kit as described in [[http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2014-3-

11#3._ChargeSwitch.C2.AE_gDNA_1_ml_Serum_kit]] for 700ul sample volume

Project ID Sample ID Plasma Volumn (ul) PBS to 700ul (ul)
UCSD-006.1 6-P_1 430.00 270.00
UCSD-006.2 6-P_2 540.00 160.00
UCSD-006.3 6-P_3 220.00 480.00
UCSD-006.4 6-P_4 550.00 150.00
UCSD-006.5 6-P_5 665.00 35.00
UCSD-006.6 6-P_6 650.00 50.00
UCSD-006.7 6-P_7 700.00 0.00
UCSD-006.8 6-P_8 690.00 10.00
UCSD-006.9 6-P_9 520.00 180.00
UCSD-006.10 6-P_10 700.00 0.00

Prep (for 700ul sample volume)

Project ID Sample ID Plasma Volumn (ul) PBS to 500ul (ul)
UCSD-007.1 7-P_1 475.00 25.00
UCSD-007.2 7-P_2 350.00 150.00
UCSD-007.3 7-P_3 400.00 100.00
UCSD-007.4 7-P_4 325.00 175.00
UCSD-007.5 7-P_5 440.00 60.00
UCSD-007.6 7-P_6 345.00 155.00
UCSD-007.7 7-P_7 320.00 180.00
UCSD-007.8 7-P_8 303.00 197.00
UCSD-007.9 7-P_9 330.00 170.00
UCSD-007.10 7-P_10 500.00 0.00

Prep (for 500ul sample volume)

  • 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 Yield (ng)
6-P_1 2 ng/mL 2 100 0.200 30.00 6.00
6-P_2 11 ng/mL 2 100 1.100 30.00 33.00
6-P_3 15.7 ng/mL 2 100 1.570 30.00 47.10
6-P_4 8.89 ng/mL 2 100 0.889 30.00 26.67
6-P_5 4.87 ng/mL 2 100 0.487 30.00 14.61
6-P_6 2.49 ng/mL 2 100 0.249 30.00 7.47
6-P_7 2.47 ng/mL 2 100 0.247 30.00 7.41
6-P_8 2.26 ng/mL 2 100 0.226 30.00 6.78
6-P_9 4.08 ng/mL 2 100 0.408 30.00 12.24
6-P_10 3.33 ng/mL 2 100 0.333 30.00 9.99
Sample Conc. in the Qubit Unit uL used Dilution Sample Conc. (ng/ul) Volume Yield (ng)
7-P_1 6.92 ng/mL 2 100 0.692 30.00 20.76
7-P_2 11.4 ng/mL 2 100 1.140 30.00 34.20
7-P_3 2.08 ng/mL 2 100 0.208 30.00 6.24
7-P_4 2.02 ng/mL 2 100 0.202 30.00 6.06
7-P_5 11.1 ng/mL 2 100 1.110 30.00 33.30
7-P_6 2.33 ng/mL 2 100 0.233 30.00 6.99
7-P_7 2.29 ng/mL 2 100 0.229 30.00 6.87
7-P_8 1.67 ng/mL 2 100 0.167 30.00 5.01
7-P_9 2.31 ng/mL 2 100 0.231 30.00 6.93
7-P_10 2.03 ng/mL 2 100 0.203 30.00 6.09

Normalize DNA yield to 1mL plasma volume

Sample Yield (ng) Serum volume (ul) Normalized yield to 1mL (ng)
6-P_1 6.00 430.00 13.95
6-P_2 33.00 540.00 61.11
6-P_3 47.10 220.00 214.09
6-P_4 26.67 550.00 48.49
6-P_5 14.61 665.00 21.97
6-P_6 7.47 650.00 11.49
6-P_7 7.41 700.00 10.59
6-P_8 6.78 690.00 9.83
6-P_9 12.24 520.00 23.54
6-P_10 9.99 700.00 14.27

UCSD-006 Plasma original color

File:UCSD-006-plasma 1-10 2.jpg
  • Based on plasma color, 6-P_3 sample had very high DNA amount due to cell lysis
Sample Yield (ng) Serum volume (ul) Normalized yield to 1mL (ng)
7-P_1 20.76 475.00 43.71
7-P_2 34.20 350.00 97.71
7-P_3 6.24 400.00 15.60
7-P_4 6.06 325.00 18.65
7-P_5 33.30 440.00 75.68
7-P_6 6.99 345.00 20.26
7-P_7 6.87 320.00 21.47
7-P_8 5.01 303.00 16.53
7-P_9 6.93 330.00 21.00
7-P_10 6.09 500.00 12.18

UCSD-007 Plasma original color

File:UCSD-007-plasma 1-10 1.jpg