Noi/NOTES/2014-8-15: Difference between revisions

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= Preparation of WGBS libraries of small fragment DNA in normal control plasma samples (no DNA fragmentation step, with dAdGdCdT for end repair) =
= Preparation of WGBS libraries of small fragment DNA in normal control plasma samples (no DNA fragmentation step, with dAdGdCdT for end repair) =
[[noi:DMR220k_LabNotes|'''Back to calendar''']]
{| {{table}} border =1
{| {{table}} border =1
| align="center" style="width:80px;background:#f0f0f0;"|'''Zhang lab ID'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Zhang lab ID'''
Line 111: Line 112:
<br>
<br>
==== <u>Ligation reaction mix</u> ====  
==== <u>Ligation reaction mix</u> ====  
{| {{table}} class = wikitable
| align="center" style="width:80px;background:dodgerblue;"|'''Exp ID'''
| align="center" style="width:80px;background:magenta;"|'''TruSeq Index'''
| align="center" style="width:80px;background:dodgerblue;"|'''Exp ID'''
| align="center" style="width:80px;background:magenta;"|'''TruSeq Index'''
| align="center" style="width:80px;background:dodgerblue;"|'''Exp ID'''
| align="center" style="width:80px;background:magenta;"|'''TruSeq Index'''
| align="center" style="width:80px;background:dodgerblue;"|'''Exp ID'''
| align="center" style="width:80px;background:magenta;"|'''TruSeq Index'''
|-
| wNC-1||1||wNC-12||13||wNC-20||11||NTC_1||3
|-
| wNC-2||2||wNC-13||14||wNC-21||12||NTC_2||9
|-
| wNC-3||4||wNC-14||15||wNC-22||21||wNC-3t||23
|-
| wNC-5||5||wNC-15||16||wNC-23||22||wNC-4t||25
|-
| wNC-6||6||wNC-16||27||wNC-24||3||||
|-
| wNC-7||7||wNC-17||18||wNC-27||9||||
|-
| wNC-8||8||wNC-18||19||wNC-29||23||||
|-
| wNC-9||10||wNC-19||20||wNC-30||25||||
|}
==== <u>Ligation reaction mix</u> ====
{| {{table}} border = 1
{| {{table}} border = 1
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''Components'''
Line 127: Line 155:
|-
|-
| Total||24.00||
| Total||24.00||
|}
|}<br>
{| {{table}} border = 1
{| {{table}} border = 1
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''Components'''
Line 159: Line 187:
* Repeat all procedure exactly the same as previous experiment on 2014-08-03
* Repeat all procedure exactly the same as previous experiment on 2014-08-03
=== 1st round PCR (fix 12 cycles) ===
=== 1st round PCR (fix 12 cycles) ===
{| {{table}} border =1
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Conc'''
| align="center" style="width:80px;background:#f0f0f0;"|'''unit'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Final conc./amount'''
| align="center" style="width:60px;background:#f0f0f0;"|'''unit'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Volume (ul)'''
|-
| Bis-cvt DNA||||||||||30.00
|-
| 10X Reaction buffer||10||X||1||X||5.00
|-
| dNTP mix||10||mM||0.25||mM||1.25
|-
| TruS_F/R||10||uM||0.3||uM||1.50
|-
| 50X SYBG||50||X||0.4||X||0.40
|-
| PfuTurbo Cx||2.5||Unit/ul||1||unit||1.00
|-
| H2O||||||||||10.85
|-
| Total ||||||||||50.00
|}
:- Aliquot 20 + 30ul BIS-DNA template
:- Mix well
:: ''95C for 2min --> [95C for 20sec -> 60C for 30sec -> 72C for 1min] X 12 --> 72C for 2min''
:- Purify with 1.25X AMPure beads
:- Elute with 42ul EB Buffer
:- Continue to 2nd round PCR. No PAGE verification.
=== 2nd round PCR ===
=== Quick test ===
{| {{table}} border =1
| align="center" style="width:140px;background:#f0f0f0;"|'''Components'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Conc'''
| align="center" style="width:80px;background:#f0f0f0;"|'''unit'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Final conc.'''
| align="center" style="width:60px;background:#f0f0f0;"|'''unit'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Volume (ul)'''
| align="center" style="width:80px;background:#f0f0f0;"|'''30 rxn mix'''
|-
| Purified 1st round DNA||||||||||3.00||0.00
|-
| 5X Phusion HF buffer||5||X||1||X||2.00||60.00
|-
| dNTP mix||10||mM||0.25||mM||0.25||7.50
|-
| TruS_F/R||10||uM||0.3||uM||0.30||9.00
|-
| 50X SYBR||50||X||0.4||X||0.08||2.40
|-
| Phusion HF||2||unit/ul||||||0.10||3.00
|-
| H2O||||||||||4.27||128.10
|-
| Total||||||||||10.00||
|}
:- Aliquot 7 + 3ul DNA template
:- Mix well.
::''98C for 2min --> [98C for 10sec -> '''60C for 30sec''' -> 72C for 1min] X 8 cycles --> 72C for 2min''<br>
:- Added 2ul 6X loading dye to each strip tube & mix well
:- Load 6ul PCR/dye mix in 6% gel and run at 250 volts for 22min.
[[File:ZhangLab_2 2014-08-17 16hr 58min_test-2ndPCR_NC-8x_gel1.jpg|450px]] [[File:ZhangLab_2 2014-08-17 16hr 58min_test-2ndPCR_NC-8x_gel2.jpg|450px]]
[[File:ZhangLab_2 2014-08-17 16hr 58min_test-2ndPCR_NC-8x_gel3.jpg|430px]]
* From quick qPCR test, there are variation of the intensity of libraries size ~300bp. 8 cycles seem to be over-amplified for almost samples. There are some samples that had very faint intensity of the libraries. I need to varied PCR cycle number for some samples.
{| {{table}} border = 1
| align="center" style="width:140px;background:#f0f0f0;"|'''Components'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Conc'''
| align="center" style="width:60px;background:#f0f0f0;"|'''unit'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Final conc.'''
| align="center" style="width:60px;background:#f0f0f0;"|'''unit'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Volume (ul)'''
| align="center" style="width:80px;background:#f0f0f0;"|'''27 rxn mix'''
|-
| Purified 1st round DNA||||||||||36.00||0.00
|-
| 5X Phusion HF buffer||5||X||1||X||24.00||648.00
|-
| dNTP mix||10||mM||0.25||mM||3.00||81.00
|-
| TruS_F/R||10||uM||0.3||uM||3.60||97.20
|-
| 50X SYBR||50||X||0.4||X||0.96||25.92
|-
| Phusion HF||2||unit/ul||||||1.20||32.40
|-
| H2O||||||||||51.24||1383.48
|-
| Total||||||||||120.00||
|}
:- Aliquot 84 + 36ul DNA template
:- Mix well. Split 38ul X3
::''98C for 2min --> [98C for 10sec -> '''60C for 30sec''' -> 72C for 1min] X 7 cycles --> 72C for 2min''<br>
:- I pool ~110ul PCR product from the two replicates, purify with 2 rounds of 1X AMPure beads (fist round elute with EB buffer 60ul. 2nd round elute with 65ul EB buffer.
:- Loaded 2ul in 6% TBE gel for PAGE verification
[[File:ZhangLab_2 2014-08-18 14hr 52min_PQ_wgbs_NC-2ul_gel1.jpg| 450px]]  [[File:ZhangLab_2 2014-08-18 14hr 52min_PQ_wgbs_NC-2ul_gel2.jpg| 450px]]
[[File:ZhangLab_2 2014-08-18 14hr 52min_PQ_wgbs_NC-2ul_gel3.jpg| 320px]]
* Different from WGBS from cancer patient small fragment DNA, there are high variations of library amount among different samples as mentioned from quick qPCR. Also, the last two samples (NC-3 and NC-4) ligated with T4 DNA ligase left at RT almost 2 days had comparable result compare to the same sample ligated with proper stored T4 DNA ligase.
=== Qubit dsDNA HS quantification of WGBS libraries from normal control cfDNA ===
{| {{table}} border =1
| align="center" style="width:80px;background:#f0f0f0;"|'''Sample'''
| align="center" style="width:80px;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:60px;background:#f0f0f0;"|'''Dilution'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Volume (ul)'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Total amount (ng)'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Used for prev. exp (ng)'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Amount left (ng)'''
| align="center" style="width:80px;background:#f0f0f0;"|'''> 400ng'''
|-
| NC-1||4.1||ng/mL||2||100||0.41||80.00||32.80||0.00||32.80||NO
|-
| NC-2||13.9||ng/mL||2||100||1.39||80.00||111.20||0.00||111.20||NO
|-
| NC-3||18||ng/mL||2||100||1.80||80.00||144.00||0.00||144.00||NO
|-
| NC-5||4.71||ng/mL||2||100||0.47||80.00||37.68||0.00||37.68||NO
|-
| NC-6||42.5||ng/mL||2||100||4.25||80.00||340.00||100.00||240.00||NO
|-
| NC-7||4.69||ng/mL||2||100||0.47||80.00||37.52||0.00||37.52||NO
|-
| NC-8||15.2||ng/mL||2||100||1.52||80.00||121.60||0.00||121.60||NO
|-
| NC-9||37.2||ng/mL||2||100||3.72||80.00||297.60||100.00||197.60||NO
|-
| NC-12||29.9||ng/mL||2||100||2.99||80.00||239.20||0.00||239.20||NO
|-
| NC-13||16.4||ng/mL||2||100||1.64||80.00||131.20||0.00||131.20||NO
|-
| NC-14||12.8||ng/mL||2||100||1.28||80.00||102.40||0.00||102.40||NO
|-
| NC-15||12.9||ng/mL||2||100||1.29||80.00||103.20||0.00||103.20||NO
|-
| NC-16||13.3||ng/mL||2||100||1.33||80.00||106.40||0.00||106.40||NO
|-
| NC-17||38.6||ng/mL||2||100||3.86||80.00||308.80||0.00||308.80||NO
|-
| NC-18||26.3||ng/mL||2||100||2.63||80.00||210.40||0.00||210.40||NO
|-
| NC-19||4.74||ng/mL||2||100||0.47||80.00||37.92||0.00||37.92||NO
|-
| NC-20||3.96||ng/mL||2||100||0.40||80.00||31.68||0.00||31.68||NO
|-
| NC-21||4.13||ng/mL||2||100||0.41||80.00||33.04||0.00||33.04||NO
|-
| NC-22||27.1||ng/mL||2||100||2.71||80.00||216.80||0.00||216.80||NO
|-
| NC-23||17.6||ng/mL||2||100||1.76||80.00||140.80||0.00||140.80||NO
|-
| NC-24||3.87||ng/mL||2||100||0.39||80.00||30.96||0.00||30.96||NO
|-
| NC-27||68.8||ng/mL||2||100||6.88||80.00||550.40||100.00||450.40||YES
|-
| NC-29||35.9||ng/mL||2||100||3.59||80.00||287.20||0.00||287.20||NO
|-
| NC-30||64.6||ng/mL||2||100||6.46||80.00||516.80||100.00||416.80||YES
|}

Latest revision as of 18:14, 17 September 2014

Preparation of WGBS libraries of small fragment DNA in normal control plasma samples (no DNA fragmentation step, with dAdGdCdT for end repair)[edit]

Back to calendar

Zhang lab ID Conc. (ng/ul) Volume for 1ng H2O # in strip Exp ID Klenow,exo-:dAdCdGdT Mix Total
NC-1 0.293 3.41 6.59 #1_1 wNC-1 10.00 20.00
NC-2 0.325 3.08 6.92 #1_2 wNC-2 10.00 20.00
NC-3 0.538 1.86 8.14 #1_3 wNC-3 10.00 20.00
NC-5 0.156 6.41 3.59 #1_4 wNC-5 10.00 20.00
NC-6 0.148 6.76 3.24 #1_5 wNC-6 10.00 20.00
NC-7 0.245 4.08 5.92 #1_6 wNC-7 10.00 20.00
NC-8 0.141 7.09 2.91 #1_7 wNC-8 10.00 20.00
NC-9 0.171 5.85 4.15 #1_8 wNC-9 10.00 20.00
NC-12 0.163 6.13 3.87 #2_1 wNC-12 10.00 20.00
NC-13 0.168 5.95 4.05 #2_2 wNC-13 10.00 20.00
NC-14 0.157 6.37 3.63 #2_3 wNC-14 10.00 20.00
NC-15 0.118 8.47 1.53 #2_4 wNC-15 10.00 20.00
NC-16 0.140 7.14 2.86 #2_5 wNC-16 10.00 20.00
NC-17 0.126 7.94 2.06 #2_6 wNC-17 10.00 20.00
NC-18 0.189 5.29 4.71 #2_7 wNC-18 10.00 20.00
NC-19 0.105 9.52 0.48 #2_8 wNC-19 10.00 20.00
NC-20 0.128 7.81 2.19 #3_1 wNC-20 10.00 20.00
NC-21 0.205 4.88 5.12 #3_2 wNC-21 10.00 20.00
NC-22 0.180 5.56 4.44 #3_3 wNC-22 10.00 20.00
NC-23 0.153 6.54 3.46 #3_4 wNC-23 10.00 20.00
NC-24 0.140 7.14 2.86 #3_5 wNC-24 10.00 20.00
NC-27 0.124 8.06 1.94 #3_6 wNC-27 10.00 20.00
NC-29 0.109 9.17 0.83 #3_7 wNC-29 10.00 20.00
NC-30 0.099 10.00 0.00 #3_8 wNC-30 10.00 20.00
NTC_1 0 0.00 10.00 #4_1 10.00 20.00
NTC_2 0 0.00 10.00 #4_2 10.00 20.00
NC-3 0.538 1.86 8.14 #4_3 wNC-3t 10.00 20.00
NC-4 0.848 1.18 8.82 #4_4 wNC-4t 10.00 20.00
  • The last two samples, NC-3 and NC-4 were used to tested for T4 DNA ligase HC that has been delivered during weekend (Saturday) and was left at RT until Monday. I got the new tube from Thermoscientific for replacement. All samples except the last two were prepared with the new tube of T4 DNA ligase HC.
  • Prepare new tube of dA:dC:dG:dT (20:2:2:2 mM)
Components Final conc. (nM) Volume (ul)
100mM dATP 20 100
100mM dCTP 2 10
100mM dGTC 2 10
100mM dTTC 2 10
H2O 370

Klenow exo- and dA:dG:dC reaction mix[edit]

Components 1x rxn 30x rxn
10X Tango buffer 2.00 60.00
dA:dC:dG:dT (20:2:2:2mM) 1.00 30.00
Klenow fragment, exo- (5U/ul) 1.00 30.00
H2O 6.00 180.00
Total 10.00

- Aliquot 32 of Klwnow, exo- and dA:dC:dG:dT reaction mix to 8-tube strip

- Add H2O to each tube
- Add 1ng of DNA to each tube
- Add 10ul of Klwnow, exo- and dA:dC:dG reaction mix to each tube of DNA with multichannel pipette. Pipette few times
- Spin down the tube
- Mix by gentle pulse-vortexing on PCR rack 10x
- Spin down the tube
- 30C, 20min --> 37C, 20min --> hold at 4C (no heat lid)
- 75C, 10min --> hold at 4C (heat lid on)
  • No AMPure bead purification


Ligation reaction mix[edit]

Exp ID TruSeq Index Exp ID TruSeq Index Exp ID TruSeq Index Exp ID TruSeq Index
wNC-1 1 wNC-12 13 wNC-20 11 NTC_1 3
wNC-2 2 wNC-13 14 wNC-21 12 NTC_2 9
wNC-3 4 wNC-14 15 wNC-22 21 wNC-3t 23
wNC-5 5 wNC-15 16 wNC-23 22 wNC-4t 25
wNC-6 6 wNC-16 27 wNC-24 3
wNC-7 7 wNC-17 18 wNC-27 9
wNC-8 8 wNC-18 19 wNC-29 23
wNC-9 10 wNC-19 20 wNC-30 25

Ligation reaction mix[edit]

Components 1x rxn 28x rxn
dA-tailed DNA 20.00 0.00
10X Tango buffer 0.50 14.00
HC T4 DNA ligase (30units/ul) 1.00 28.00
10mM ATP 1.25 35.00
H2O 1.25 35.00
Total 24.00


Components 1x rxn 2x rxn
dA-tailed DNA 20.00 0.00
10X Tango buffer 0.50 1.00
THAWED HC T4 DNA ligase (30units/ul) 1.00 2.00
10mM ATP 1.25 2.50
H2O 1.25 2.50
Total 24.00
- Aliquot 13ul of ligation reaction mix to 8-tube strip
- Add 1ul of 1:20 diluted TruSeq adaptors and pipette few times to make sure adaptors were added to the reaction
- Add 4ul of ligation reaction mix with multichannel pipette
- Spin down the tube
- Mix by gentle pulse-vortexing on PCR rack 10x
- Spin down the tube
- 16C, 20h (no heat lid)
- Heat inactivate at 65C, 20min --> hold at 4C (heat lid on)
- Continue to bisulfite conversion

Bisulfite conversion[edit]

Bisulfite conversion procedures

Amplification[edit]

  • Repeat all procedure exactly the same as previous experiment on 2014-08-03

1st round PCR (fix 12 cycles)[edit]

Components Conc unit Final conc./amount unit Volume (ul)
Bis-cvt DNA 30.00
10X Reaction buffer 10 X 1 X 5.00
dNTP mix 10 mM 0.25 mM 1.25
TruS_F/R 10 uM 0.3 uM 1.50
50X SYBG 50 X 0.4 X 0.40
PfuTurbo Cx 2.5 Unit/ul 1 unit 1.00
H2O 10.85
Total 50.00
- Aliquot 20 + 30ul BIS-DNA template
- Mix well
95C for 2min --> [95C for 20sec -> 60C for 30sec -> 72C for 1min] X 12 --> 72C for 2min
- Purify with 1.25X AMPure beads
- Elute with 42ul EB Buffer
- Continue to 2nd round PCR. No PAGE verification.

2nd round PCR[edit]

Quick test[edit]

Components Conc unit Final conc. unit Volume (ul) 30 rxn mix
Purified 1st round DNA 3.00 0.00
5X Phusion HF buffer 5 X 1 X 2.00 60.00
dNTP mix 10 mM 0.25 mM 0.25 7.50
TruS_F/R 10 uM 0.3 uM 0.30 9.00
50X SYBR 50 X 0.4 X 0.08 2.40
Phusion HF 2 unit/ul 0.10 3.00
H2O 4.27 128.10
Total 10.00
- Aliquot 7 + 3ul DNA template
- Mix well.
98C for 2min --> [98C for 10sec -> 60C for 30sec -> 72C for 1min] X 8 cycles --> 72C for 2min
- Added 2ul 6X loading dye to each strip tube & mix well
- Load 6ul PCR/dye mix in 6% gel and run at 250 volts for 22min.
File:ZhangLab 2 2014-08-17 16hr 58min test-2ndPCR NC-8x gel1.jpg File:ZhangLab 2 2014-08-17 16hr 58min test-2ndPCR NC-8x gel2.jpg

File:ZhangLab 2 2014-08-17 16hr 58min test-2ndPCR NC-8x gel3.jpg
  • From quick qPCR test, there are variation of the intensity of libraries size ~300bp. 8 cycles seem to be over-amplified for almost samples. There are some samples that had very faint intensity of the libraries. I need to varied PCR cycle number for some samples.
Components Conc unit Final conc. unit Volume (ul) 27 rxn mix
Purified 1st round DNA 36.00 0.00
5X Phusion HF buffer 5 X 1 X 24.00 648.00
dNTP mix 10 mM 0.25 mM 3.00 81.00
TruS_F/R 10 uM 0.3 uM 3.60 97.20
50X SYBR 50 X 0.4 X 0.96 25.92
Phusion HF 2 unit/ul 1.20 32.40
H2O 51.24 1383.48
Total 120.00
- Aliquot 84 + 36ul DNA template
- Mix well. Split 38ul X3
98C for 2min --> [98C for 10sec -> 60C for 30sec -> 72C for 1min] X 7 cycles --> 72C for 2min
- I pool ~110ul PCR product from the two replicates, purify with 2 rounds of 1X AMPure beads (fist round elute with EB buffer 60ul. 2nd round elute with 65ul EB buffer.
- Loaded 2ul in 6% TBE gel for PAGE verification
File:ZhangLab 2 2014-08-18 14hr 52min PQ wgbs NC-2ul gel1.jpg  File:ZhangLab 2 2014-08-18 14hr 52min PQ wgbs NC-2ul gel2.jpg

File:ZhangLab 2 2014-08-18 14hr 52min PQ wgbs NC-2ul gel3.jpg
  • Different from WGBS from cancer patient small fragment DNA, there are high variations of library amount among different samples as mentioned from quick qPCR. Also, the last two samples (NC-3 and NC-4) ligated with T4 DNA ligase left at RT almost 2 days had comparable result compare to the same sample ligated with proper stored T4 DNA ligase.

Qubit dsDNA HS quantification of WGBS libraries from normal control cfDNA[edit]

Sample Conc. in the Qubit Unit uL used Dilution Conc. (ng/ul) Volume (ul) Total amount (ng) Used for prev. exp (ng) Amount left (ng) > 400ng
NC-1 4.1 ng/mL 2 100 0.41 80.00 32.80 0.00 32.80 NO
NC-2 13.9 ng/mL 2 100 1.39 80.00 111.20 0.00 111.20 NO
NC-3 18 ng/mL 2 100 1.80 80.00 144.00 0.00 144.00 NO
NC-5 4.71 ng/mL 2 100 0.47 80.00 37.68 0.00 37.68 NO
NC-6 42.5 ng/mL 2 100 4.25 80.00 340.00 100.00 240.00 NO
NC-7 4.69 ng/mL 2 100 0.47 80.00 37.52 0.00 37.52 NO
NC-8 15.2 ng/mL 2 100 1.52 80.00 121.60 0.00 121.60 NO
NC-9 37.2 ng/mL 2 100 3.72 80.00 297.60 100.00 197.60 NO
NC-12 29.9 ng/mL 2 100 2.99 80.00 239.20 0.00 239.20 NO
NC-13 16.4 ng/mL 2 100 1.64 80.00 131.20 0.00 131.20 NO
NC-14 12.8 ng/mL 2 100 1.28 80.00 102.40 0.00 102.40 NO
NC-15 12.9 ng/mL 2 100 1.29 80.00 103.20 0.00 103.20 NO
NC-16 13.3 ng/mL 2 100 1.33 80.00 106.40 0.00 106.40 NO
NC-17 38.6 ng/mL 2 100 3.86 80.00 308.80 0.00 308.80 NO
NC-18 26.3 ng/mL 2 100 2.63 80.00 210.40 0.00 210.40 NO
NC-19 4.74 ng/mL 2 100 0.47 80.00 37.92 0.00 37.92 NO
NC-20 3.96 ng/mL 2 100 0.40 80.00 31.68 0.00 31.68 NO
NC-21 4.13 ng/mL 2 100 0.41 80.00 33.04 0.00 33.04 NO
NC-22 27.1 ng/mL 2 100 2.71 80.00 216.80 0.00 216.80 NO
NC-23 17.6 ng/mL 2 100 1.76 80.00 140.80 0.00 140.80 NO
NC-24 3.87 ng/mL 2 100 0.39 80.00 30.96 0.00 30.96 NO
NC-27 68.8 ng/mL 2 100 6.88 80.00 550.40 100.00 450.40 YES
NC-29 35.9 ng/mL 2 100 3.59 80.00 287.20 0.00 287.20 NO
NC-30 64.6 ng/mL 2 100 6.46 80.00 516.80 100.00 416.80 YES