多重基因表达.ppt

Gene Panel target gene Product Size w/Universals A_Gene10 149 A_Gene17 156 A_Gene5 161 A_Gene13 175 A_Gene7 183 A_Gene2 189 A_Gene21 196 A_Gene15 203 A_Gene12 211 A_Gene8 224 A_Gene19 238 A_Gene20 245 A_Gene4 252 A_Gene1 259 A_Gene6 273 AT1G49240 280 A_Gene3 288 A_Gene9 293 A_Gene18 302 A_Gene14 310 A_Gene16 334 A_Gene11 350 Sample Preparation Total RNA isolation using Agencourt RNAPrep?kit RT-PCR using GenomeLab GeXP Rat Multitox Plex kit Rat p rimary hepatocytes Rat liver cell line (C9) Cells Treated with Chemicals or Vehicle Controls in 96 Well Tissue Culture Plates Breast Cancer Study (SBS 06) Breast Cancer Study (SBS 06) 实 例 讲 解 ? Design primer 1 Design primer 2 Reverse transcription Reagent RT(- )(μL ) NTC (μL) Sample (μL) 5XRT buffer 2 2 2 KanR RNA 2.5 2.5 2.5 DNase/RNase Free H 2 O 3.5 4 3 RT primer 1 1 1 RT enzyme - 0.5 0.5 Sample RNA 1 - 1 Total Volum 10 * RT reaction was performed under the conditions : 1 min at 48 ° C, 60 min at 42 ° C, 5 min at 95 ° C, and hold at 4 ° C. Polymerase chain reaction PCR Reaction Mix Volume per well 5X PCR buffer 2 25mM MgCL2 2 Thermo-Start DNA Polymerase 0.35 PCR primer Mix 1 RT Product 4.5 Total Volume 9.85 * PCR reaction was performed under the conditions : 10 min at 95 ° C, followed by 40 cycles of 30 s at 94 ° C , 30 s at 55 ° C, and 1 min at 68 ° C; hold at 4 ° C. Single Reaction Kan R Kan R Kan R 1β β -actin IL- 1β Single Reaction Kan R Kan R Kan R Kan R GK IL-8 IFN- г ubiquitin Optimization of Reverse Primer Reference Peak -0 2 5 0 0 5 0 0 0 7 5 0 0 1 0 0 0 0 1 2 5 0 0 1 5 0 0 0 1 7 5 0 0 2 0 0 0 0 2 2 5 0 0 1 2 5 1 5 0 1 7 5 2 0 0 2 2 5 2 5 0 2 7 5 3 0 0 3 2 5 3 5 0 1 0 - 1 1 - 1 8 - 1 0 - 1 1 - 1 8 - P M - K 3 - j x w - -.G 1 1 _ 1 0 1 1 1 8 2 2 0 H S iz e (n t) 139.03 147.42 150.18 157.40 173.35 187.82 193.00 201.01 209.19 213.22 229.11 231.84 260.30 288.8