Sample Size R02548045 蘇宸瑩 D02548002 蔡靜雯 2014.12.03.

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Presentation transcript:

Sample Size R 蘇宸瑩 D 蔡靜雯

 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 )  Sample preparation a) an extract of the test sample b) 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/cm 2 )  n = 4  ** p < 0.01 (Student’s t-test) **

 Experiment I & II (N=2)  Problem: How do we analyze these data for significant results? **

 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 gene E : Endogenous gene (internal control) S : Sample Ctrl : Control c- MycGAPDHΔ CtΔ Δ Ct T = T = 12 hr T = 24 hr T = 48 hr

 Experiment I & II (N=2)  Problem: How do we analyze these data for significant results? **

controlAB ODcellsOD /cellsODcellsOD /cellsODcellsOD /cells E E E E E E E E E E E E-06 Average 6.212E E E-06 exp./control controlAB ODcellsOD /cellsODcellsOD /cellsODcellsOD /cells E E E E E E E E E E E E-06 Average 4.217E E E-06 exp./control Experiment I Experiment II

Experiment I Experiment II Experiment I + II