Noi/NOTES/2014-6-27

From ZhangLabWiki
Revision as of 19:20, 28 June 2014 by >Noi
Jump to navigation Jump to search

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 [[1]] for 600ul sample volume

Prep