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= DNA extraction of 10 plasma samples and 5 matched pair solid tumor tissue samples from Cancer Center = * [[http://genome-tech.ucsd.edu/LabNotes/index.php/noi:DMR220k_LabNotes '''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. === <span style="color:blue"><u>Part 1: DNA extraction from plasma samples</u></span> === * There are <u>three tubes</u> 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 :{| {{table}} class = wikitable | align="center" style=""width:120px;background:#f0f0f0;"|'''Project ID #''' | align="center" style=""width:120px;background:#f0f0f0;"|'''Zhang lab ID''' | align="center" style=""width:100px;background:#f0f0f0;"|'''Volume 2 tubes''' | align="center" style=""width:100px;background:#f0f0f0;"|'''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 [[http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2014-3-11#3._ChargeSwitch.C2.AE_gDNA_1_ml_Serum_kit]] for 600ul sample volume '''''<span style="color:crimson"><u>Prep</u></span>'''''<br> * 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 ==== {| {{table}} class = wikitable | align="center" style="width:120px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:100px;background:#f0f0f0;"|'''Conc. in the Qubit''' | align="center" style="width:60px;background:#f0f0f0;"|'''Unit''' | align="center" style="width:60px;background:#f0f0f0;"|'''uL used''' | align="center" style="width:70px;background:#f0f0f0;"|'''Dilution''' | align="center" style="width:70px;background:#f0f0f0;"|'''Sample conc. (ng/uL)''' | align="center" style="width:70px;background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="width:70px;background:#f0f0f0;"|'''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 ==== {| {{table}} class = wikitable | align="center" style="width:120px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:100px;background:#f0f0f0;"|'''Yields (ng)''' | align="center" style="width:100px;background:#f0f0f0;"|'''Plasma volume (ul)''' | align="center" style="width:100px;background:#f0f0f0;"|'''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|500px]] === <span style="color:blue"><u>Part 2: DNA extraction from solid tumor tissue samples</u></span> === * 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: [[http://mvz.berkeley.edu/egl/inserts/DNeasy_Blood_&_Tissue_Handbook.pdf]]. 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 ==== {| {{table}} class = wikitable | align="center" style="width:120px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:100px;background:#f0f0f0;"|'''Conc. In the Qubit''' | align="center" style="width:60px;background:#f0f0f0;"|'''Unit''' | align="center" style="width:60px;background:#f0f0f0;"|'''uL used''' | align="center" style="width:70px;background:#f0f0f0;"|'''Dilution''' | align="center" style="width:70px;background:#f0f0f0;"|'''Sample conc. (ng/ul)''' | align="center" style="width:70px;background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="width:70px;background:#f0f0f0;"|'''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 {| {{table}} class = wikitable | align="center" style="width:120px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:100px;background:#f0f0f0;"|'''ND conc. (ng/ul)''' | align="center" style="width:100px;background:#f0f0f0;"|'''260/280''' | align="center" style="width:100px;background:#f0f0f0;"|'''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.
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