多重基因表达.pptVIP

Gene Panel Sample Preparation Total RNA isolation using Agencourt RNAPrep?kit RT-PCR using GenomeLab GeXP Rat MultitoxPlex kit Rat primary 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 H2O 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 Reference Peak Before Optimize the Reverse Primer Kan R Optimization of Reverse Primer After Dilute the four Reverse Primers Reference Peak Kan R Optimization of Reverse Primer After Double the Four Reverse Primers Reference Peak 19 peaks = 18 genes + internal control KanR Kan R Optimization of Reverse Primer Add another Two Reverse Primers Reference Peak ADD1 PECAM-1 Precision experiment assessment for GeXP analyzer Within- run precision ( 66 ng evel ) Precision experiment assessment for GeXP analyzer Within- run precision ( 33 ng evel ) Precision experiment assessment for GeXP analyzer Bwtween - run precision ( 66 ng evel ) FR：有问题，需排除原因。 ND: 未检测到。 经验总结 目的片段的设计：由于产物通过毛细管电泳通过设计片段长度区分，因此目的片段间隔越大，分离效果越好，本研究设计的目的片段长度均在3个nt以上，有效保证了对片段的分离； 多引物影响：体系中存在多对PCR引物，引物间存在相互作用，在