1. Franck CHATELAIN Q-PCR on DICER project and PKD project Animal facility Myography on PKD project Ingénieur d’études

2. Does the expression of PKD1 in smooth muscle control the expression of other genes ? How ? Where ? Using quantitative PCR, we would like to know if the deletion, and by extension, the expression of PKD1 has an impact on the expression of : - PKD2 - other PKDs - Metalloproteinases (MMPs) - Metalloproteinase inhibitors (TIMPs) - other channels. - Heart - Aortas - Cerebral arteries

3. Primers with good parameters for Q-PCR As we need a relative large amount of RNA to generate the cDNA, we set up the Q-PCR parameters on big organs like Heart, Kidney, Brain, Testis before testing on aortas and cerebral arteries So far we have found the good parameters for the following oligos : - PKD1 - PKD2 - MMP2 - MMP9 - Fibrocystin - Nephrocystin Good parameters: Tm unique and close to the theorical Tm 14<<CT<<36 Efficiency = 1.984 Efficiency  same as housekeeping primers  2

4. Finding the good couple of primers for Q-PCR Bad parameters: Multiple Tm and/or  theorical Tm CT<<14 or 36<<CT Efficiency = 1.176 Efficiency  same as housekeeping primers  2 We still need to find the right couple of primers for: - PKD1L1 - PKD1L2 - PKD1L3 - PKD2L1 - PKD2L2 - PKD-Rej - TIMP1

5. Example of Q-PCR experiments on Dicer project CT Housekee ping gene TOP1 DICERDGCR8DroshaP68P72 CTs measured Wild type Mouse 22.7534.326.8524.22127.8 Knocked Out 21.734.625.8723.119.927  CT vs TOP1 Wild type Mouse 00000 Knocked Out 1.350.070-0.010.25 2 -  CT Wild type Mouse 11111 Knocked Out 0.390.9511.010.84