Noi/NOTES/2011-3-30
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[[1]]
Verification of DMR.S1-S5 and DMR.Exp1-3 probe set quality and concentration by PAGE quantification[edit]
File:ZhangLab 2 2011-03-30 13hr 06min Testprobe-1.jpg File:ZhangLab 2 2011-03-30 13hr 10min Test probe2.jpg
Probe preparation[edit]
- Dilute each probe to final concentration 2ng/ul based on original concentration
- Mix 10ul of 2ng/ul probe with 10ul 2XTBU loading buffer
- Mix well, heat at 75C for 8min, quick cook on ice for 3min
- Load 6 and 3 ul in pre-run 6% TBU PAGE gel
- Quantify concentration and calculate back to final concentration
- Compare value of new quantification with original concentration
DMR220k Probe set | Original conc. (ng/ul) | New conc. (ng/ul) | % different |
DMR.S1 (undiluted) | 26.20 | 30.82 | 14.99 |
DMR.S2 (undiluted) | 30.10 | 40.38 | 25.46 |
DMR.S3 | 23.20 | 29.28 | 20.77 |
DMR.S4 | 24.80 | 26.94 | 7.94 |
DMR.S5 | 24.70 | 25.68 | 3.82 |
Exp1 | 33.00 | 31.88 | 3.50 |
Exp2 | 25.00 | 24.11 | 3.68 |
Exp3 | 29.00 | 28.90 | 0.35 |
DMR.S1 (diluted) | 2.00 | 1.86 | 7.63 |
DMR.S2 (diluted) | 2.00 | 1.95 | 2.67 |
Bisulfite conversion of JURKAT gDNA[edit]
Quantify bis-cvt JURKAT gDNA concentration by Qubit ssDNA assay[edit]
Sample | Concentration in the Qubit | ' | uL used | Dilution | Sample Concentration | ' | final conc (ng/ul) | Total yields in 17ul (ng) |
JURKAT 1 | 416.00 | ng/mL | 1.00 | 200.00 | 416.00 | ng/mL | 83.20 | 1414.40 |
JURKAT 2 | 417.00 | ng/mL | 1.00 | 200.00 | 417.00 | ng/mL | 83.40 | 1417.80 |
JURKAT 3 | 365.00 | ng/mL | 1.00 | 200.00 | 365.00 | ng/mL | 73.00 | 1241.00 |
JURKAT 4 | 358.00 | ng/mL | 1.00 | 200.00 | 358.00 | ng/mL | 71.60 | 1217.20 |
Ave conc. | 77.80 | 1322.60 |
Capture set up[edit]
Capture set | Details |
A | MS1. DMR.S1-3 mix multiplier C |
B | MS2. DMR.S1-3 mix multipier D |
C | ME1. DMR.Exp1-3 mix |
D | S1 100:1 ratio |
E | S2 100:1 ratio |
F | S3 100:1 ratio |
G | Exp1 100:1 ratio |
H | Exp2 100:1 ratio |
I | Exp3 100:1 ratio |
DMR220k Probe set | Conc. (ng/ul) |
DMR.S1 (undiluted) | 26.20 |
DMR.S2 (undiluted) | 30.10 |
DMR.S3 | 23.20 |
DMR.S4 | 24.80 |
DMR.S5 | 24.70 |
Exp1 | 31.88 |
Exp2 | 24.11 |
Exp3 | 28.90 |
DMR.S1 (diluted) | 1.86 |
DMR.S2 (diluted) | 1.95 |
- Note: Still use the original concentration of some probe set since the new concentration look overestimated.
A. MS1. DMR.S1-3 mix multiplier C[edit]
- Note: Multiplier C of S1-S3 was used for PD brain samples
Probes | Amount probe required 100:1 ratio (ng) |
Multiplier | Amount probes required x multiplier (ng) |
Conc(ng/ul) | Volume (ul) | ' | ' | ' | ' |
DMR220k.S1 | 2.23 | 0.05 | 0.11 | 0.10 | 1.12 | ||||
DMR220k.S2 | 2.23 | 0.09 | 0.20 | 0.10 | 2.01 | ||||
DMR220k.S3 | 18.90 | 0.25 | 4.73 | 2.00 | 2.36 | B+OS mix | bis-cvt JURKAT | H2O | Total volume |
total volume | 5.48 | 3.00 | 2.60 | 8.92 | 20.00 |
B. MS2. DMR.S1-3 mix multipier D[edit]
- Note: Multiplier D of S1-S3 was used for Guanghui's samples (Belmonte's lab)
Probes | Amount probe required 100:1 ratio (ng) |
Multiplier | Amount probes required x multiplier (ng) |
Conc(ng/ul) | Volume (ul) | ' | ' | ' | ' |
DMR220k.S1 | 2.23 | 0.56 | 1.25 | 1.86 | 0.67 | ||||
DMR220k.S2 | 2.23 | 1.00 | 2.23 | 1.95 | 1.14 | ||||
DMR220k.S3 | 18.90 | 2.78 | 52.54 | 23.20 | 2.26 | B+OS mix | bis-cvt JURKAT | H2O | Total volume |
total volume | 4.08 | 3.00 | 2.60 | 10.32 | 20.00 |
C. ME1. DMR.Exp1-3 mix[edit]
Probes | Amount probe required 100:1 ratio (ng) |
Multiplier | Amount probes required x multiplier (ng) |
Conc(ng/ul) | Volume (ul) | ' | ' | ' | ' |
DMR220k.Exp1 | 18.90 | 1.00 | 18.90 | 31.88 | 0.59 | ||||
DMR220k.Exp2 | 18.90 | 1.00 | 18.90 | 24.11 | 0.78 | ||||
DMR220k.Exp3 | 18.90 | 1.00 | 18.90 | 28.90 | 0.65 | B+OS mix | bis-cvt JURKAT | H2O | Total volume |
total volume | 2.03 | 3.00 | 2.60 | 12.37 | 20.00 |
Group | Probes | Amount probe required 100:1 ratio (ng) |
Multiplier | Amount probes required x multiplier (ng) |
Conc(ng/ul) | Volume (ul) | B+OS mix | bis-cvt JURKAT | H2O | Total volume |
D S1 | DMR220k.S1 | 2.23 | 1.00 | 2.23 | 1.86 | 1.20 | 3.00 | 2.60 | 13.20 | 20.00 |
E S2 | DMR220k.S2 | 2.23 | 1.00 | 2.23 | 1.95 | 1.14 | 3.00 | 2.60 | 13.26 | 20.00 |
F S3 | DMR220k.S3 | 18.90 | 1.00 | 18.90 | 23.20 | 0.81 | 3.00 | 2.60 | 13.59 | 20.00 |
G Exp1 | DMR220k.Exp1 | 18.90 | 1.00 | 18.90 | 31.88 | 0.59 | 3.00 | 2.60 | 13.81 | 20.00 |
H Exp2 | DMR220k.Exp2 | 18.90 | 1.00 | 18.90 | 24.11 | 0.78 | 3.00 | 2.60 | 13.62 | 20.00 |
I Exp3 | DMR220k.Exp3 | 18.90 | 1.00 | 18.90 | 28.90 | 0.65 | 3.00 | 2.60 | 13.75 | 20.00 |
Reaction mix[edit]
Components | 1rxn (ul) | 11 rxn |
10X AmpLigase Buffer | 2.00 | 22.00 |
105nM Oligo suppressor | 1.00 | 11.00 |
Total volume (ul) | 3.00 | aliquot 3ul to each capture tube |
- Note:
- B = AmpLigase buffer
- OS = Oligo suppressor
Program
-> 95c 30sec -> cool down to 55C at 0.02C/sec -> 55C 20h -> add 2ul SLN mix(2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 100uM dNTP) -> 55C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold.
Q-PCR[edit]
- do triplicate for each sample
Components | 1rxn | 29x rxn mix |
Template | 3.00 | 0.00 |
100uM AmpF6.3 | 0.10 | 2.90 |
100uM AmpR6.3 | 0.10 | 2.90 |
Phusion HF 2x MM | 25.00 | 725.00 |
50x SYBR Green | 0.40 | 11.60 |
H2O | 21.40 | 620.60 |
Total | 50.00 | aliquot 47ul, add 3ul template |
Program
98C 30sec (98C 10sec -> 58C 20sec-> 72C 20sec) x 21 cycles 72C 3min 15C hold.
Q-PCR Result[edit]
Probe set | Ct Mean | Ct Dev. | Ratio compared to s1 | Probes size | Adjusted Ratio | % of s1 |
S1-3 MultiC | 17.83 | 0.34 | 1.17 | 68000 | 0.11 | 11.21% |
S1-3 MultiD | 15.54 | 0.14 | 5.74 | 68000 | 0.55 | 54.83% |
Exp1-3mix | 15.10 | 0.27 | 7.78 | 165000 | 0.31 | 30.65% |
S1 100:1 | 18.06 | 0.19 | 1.00 | 6500 | 1.00 | 100.00% |
S2 100:1 | 18.60 | 0.27 | 0.69 | 6500 | 0.69 | 68.78% |
S3 100:1 | 16.66 | 0.20 | 2.64 | 55000 | 0.31 | 31.19% |
Exp1 100:1 | 17.21 | 0.31 | 1.80 | 55000 | 0.21 | 21.30% |
Exp2 100:1 | 16.87 | 0.02 | 2.28 | 55000 | 0.27 | 26.96% |
Exp3 100:1 | 17.39 | 0.07 | 1.59 | 55000 | 0.19 | 18.80% |
PAGE analysis[edit]
File:ZhangLab 2 2011-04-06 13hr 58min PAGE Q-PCR2ul.jpg
- Loaded sample 2ul
- Repeated Q-PCR again using the same condition by running for 18 cycles to be able to compare the band intensity of each probe set
PAGE analysis[edit]
File:ZhangLab 2 2011-04-06 17hr 16min PAGE Q-PCR 1-5ul.jpg
- Loaded sample 1.5ul
Result from PAGE quantification[edit]
Probe set | Ratio compared to s1 | Adjusted Ratio | % of s1 |
S1-3 MultiC | 0.97 | 0.09 | 9.26% |
S1-3 MultiD | 4.30 | 0.41 | 41.12% |
Exp1-3mix | 5.31 | 0.21 | 20.90% |
S1 100:1 | 1.00 | 1.00 | 100.00% |
S2 100:1 | 0.62 | 0.62 | 61.98% |
S3 100:1 | 1.75 | 0.21 | 20.67% |
Exp1 100:1 | 1.52 | 0.18 | 17.96% |
Exp2 100:1 | 2.17 | 0.26 | 25.62% |
Exp3 100:1 | 1.77 | 0.21 | 20.97% |
- A = S1-3 MultiC
- B = S1-3 MultiD
- C = Exp1-3 mix
- D = S1 100:1
- E = S2 100:2
- F = S3 100:3
- G = Exp1 100:1
- H = Exp2 100:1
- I = Exp3 100:1
- From this result, PAGE analysis supports the Q-PCR result