1. Sample Size R02548045 蘇宸瑩 D02548002 蔡靜雯 2014.12.03

2.  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

3.  Cytotoxicity (ISO 10993-5)  Sample preparation a) an extract of the test sample b) the test sample itself.

4.  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) **

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

6.  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

7.  Reverse transcription PCR  DNA→ PCR (amplify) → Gel electrophoresis

8.  Real-time PCR  Also called quantitative PCR, Q-PCR

9.  Fluoroance SYBR Green 1 dye TagMan® probe

10.  Amplification plot

11.  Relative Quantification

12.  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 = 02510150 T = 12 hr241014 T = 24 hr231112-3 T = 48 hr2810183

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

14. controlAB ODcellsOD /cellsODcellsOD /cellsODcellsOD /cells 0.601950006.328E-060.583945006.165E-060.442900004.916E-06 0.584948006.158E-060.594940006.317E-060.461880005.240E-06 0.599953006.282E-060.594952006.238E-060.479890005.387E-06 0.577949006.079E-060.581954306.085E-060.447860005.200E-06 Average 6.212E-06 6.201E-06 5.185E-06 exp./control 1 0.998 0.835 controlAB ODcellsOD /cellsODcellsOD /cellsODcellsOD /cells 0.299720004.151E-060.313700004.466E-060.265750003.532E-06 0.296725004.087E-060.318680004.674E-060.258760003.399E-06 0.318710004.477E-060.282690004.084E-060.278780003.565E-06 0.301726004.152E-060.305730004.181E-060.272750003.631E-06 Average 4.217E-06 4.351E-06 3.532E-06 exp./control 1 1.032 0.838 Experiment I Experiment II

15. Experiment I Experiment II Experiment I + II