sample size r02548045 蘇宸瑩 d02548002 蔡靜雯 2014.12.03
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Sample Size
R02548045 蘇宸瑩D02548002 蔡靜雯
2014.12.03
Biocompatibility (ISO 10993)
Part 3. Tests for genotoxicity, carcinogenicity and reproductive toxicity
Part 4. Selection of tests for interactions with blood
Part 5. Tests for in vitro cytotoxicity
Part 10. Tests for irritation and delayed-type hypersensitivity
Part 11. Tests for systemic toxicity
Cytotoxicity (ISO 10993-5)
Sample preparationa) an extract of the test sampleb) the test sample itself.
Experiment I (N=1)
PU gel
(Cross-link: A > B)
Extraction: 37°C, 24 hours
Cell: human fibroblasts
(Seeding density: 2000/cm2)
n = 4
** p < 0.01 (Student’s t-test)Control A B
Abs
orba
nce
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7 **
Experiment I & II (N=2)
Problem:
How do we analyze these data for significant results?
Control A B
Abs
orba
nce
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
Control A BA
bsor
banc
e
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35** **
PCR used to amplify a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence.
Polymerase Chain Reaction (PCR)
Denaturation
Annealing Elongation
Reverse transcription PCR
DNA→ PCR (amplify) → Gel electrophoresis
Real-time PCR
Also called quantitative PCR, Q-PCR
Fluoroance
SYBR Green 1 dye
TagMan® probe
Amplification plot
Relative Quantification
Data analysis
Δ Ct = Ct(T) – Ct(E)
ΔΔCt = ΔCt(S) – ΔCt(Ctrl)
Copy number = 2 - ΔΔCt
Example:
T : Target geneE : Endogenous gene
(internal control)S : SampleCtrl : Control
c- Myc GAPDH Δ Ct Δ Δ Ct
T = 0 25 10 15 0
T = 12 hr 24 10 14 -1
T = 24 hr 23 11 12 -3
T = 48 hr 28 10 18 3
Experiment I & II (N=2)
Problem:
How do we analyze these data for significant results?
Control A B
Abs
orba
nce
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
Control A BA
bsor
banc
e
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35** **
control A B
OD cells OD /cells OD cells OD /cells OD cells OD /cells
0.601 95000 6.328E-06 0.583 94500 6.165E-06 0.442 90000 4.916E-06
0.584 94800 6.158E-06 0.594 94000 6.317E-06 0.461 88000 5.240E-06
0.599 95300 6.282E-06 0.594 95200 6.238E-06 0.479 89000 5.387E-06
0.577 94900 6.079E-06 0.581 95430 6.085E-06 0.447 86000 5.200E-06
Average 6.212E-06 6.201E-06 5.185E-06
exp./control 1 0.998 0.835
control A B
OD cells OD /cells OD cells OD /cells OD cells OD /cells
0.299 72000 4.151E-06 0.313 70000 4.466E-06 0.265 75000 3.532E-06
0.296 72500 4.087E-06 0.318 68000 4.674E-06 0.258 76000 3.399E-06
0.318 71000 4.477E-06 0.282 69000 4.084E-06 0.278 78000 3.565E-06
0.301 72600 4.152E-06 0.305 73000 4.181E-06 0.272 75000 3.631E-06
Average 4.217E-06 4.351E-06 3.532E-06
exp./control 1 1.032 0.838
Experiment I
Experiment II
Control A B
Rel
ativ
e O
.D./c
ells
(ex
p./c
ontr
ol)
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Control A B
Rel
ativ
e O
.D./c
ells
(ex
p./c
ontr
ol)
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Control A B
Rel
ativ
e O
.D./c
ells
(ex
p./c
ontr
ol)
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Experiment I
Experiment II
Experiment I + II
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