Relative Quantifications Data Management Analysis Settings.pdf

Relative Quantification and Gene Expression Analysis 相对定量在基因表达分析中的应用 Yoland Yu Yolanda_Yu@ Field Application Specialist Bio-Rad Laboratories(Shanghai)LTD AMPLIFICATION /genomics/pcrsupport ? Assay design is the key to success ? For eukaryotes, design primers to bind to different exons or if possible over exon/exon junctions – Promotes amplification of cDNA over contaminating genomic DNA Gene Expression AMPLIFICATION /genomics/pcrsupport ? Avoid Pseudo genes amplifiaction – No-RT Control – RNase free DNase treatment ? Treat samples when possible using RNase free DNase ? Design primers for target and reference having similar properties ? Use the right reference gene(s) Gene Expression AMPLIFICATION /genomics/pcrsupport Reference Genes ? The correct selection of the reference gene for your quantitative real-time PCR reactions is essential. ? Due to the complexity of biological systems, NO single reference gene will serve the purpose of normalization for All assays. Reference gene Expression can vary with treatment, biological process and/or tissue or cell type. ? The reference gene selected must have consistent expression （“unregulated”）in the experimental system being studied. AMPLIFICATION /genomics/pcrsupport ? Test and validate reference genes in your system ? Examples : β-Actin, GAPDH, 18s rRNA, cyclophilins，IL1-b，etc. Actin Ref. Gene IL1-b Target Gene Gene Expression AMPLIFICATION /genomics/pcrsupport Reference Gene Validation 内参基因验证 ? “Normalize” input sample （处理和对照）by some means and test expression consistency – Input RNA – Cell number – Tissue weight – Input cDNA ? CT or calculated concentration of reference message should remain the same AMPLIFICATION /genomics/pcrsupport = Culture Dish Count Cells All cells plated same time from same suspension Control Quantitate RNA RT, qPCR Experimental Count Cells Quantitate RNA RT, qPCR Reference Gene Validation 内参基因验证 AMPLIFICATION /genomics/pcrsupport Reference Gene Validation Cell Number ng RNA C