实时定量PCR技术原理及应用--TIANGEN.ppt

QUALITY CONTROL -EFFICIENCY CURVES use pcr baseline subtraction (not curve fitting default option) - set the threshold manually to lab standard check all melting curves are OK check slopes are parallel in log view delete samples if multiple dilutions cross line together (usually at dilute end of curve) delete samples if can detect amplification at cycle 10 or earlier make sure there are 5 or more points check correlation coefficient is more than 0.990 Standards same copy number in all cells expressed in all cells medium copy number advantageous correction more accurate Standards The perfect standard does not exist Standards Commonly used standards Glyceraldehyde-3-phosphate dehydrogenase mRNA Beta-actin mRNA MHC I (major histocompatability complex I) mRNA Cyclophilin mRNA mRNAs for certain ribosomal proteins E.g. RPLP0 (ribosomal protein, large, P0; also known as 36B4, P0, L10E, RPPO, PRLP0, 60S acidic ribosomal protein P0, ribosomal protein L10, Arbp or acidic ribosomal phosphoprotein P0) 28S or 18S rRNA Importance of controls negative control (no DNA) checks reagents for contamination no reverse transcriptase control detects if signal from contaminating DNA positive control checks that reagents and primers work especially importance if trying to show absence of expression of a gene Standards same copy number in all cells expressed in all cells medium copy number advantageous correction more accurate reasonably large intron no pseudogene no alternate splicing in region you want to PCR Molecular beacon (分子信标) 应用范围 起始模板的定量 基因型分析 产物鉴定 SNP（单核苷酸多态性）分析 Molecular beacon (分子信标) 优缺点 高特异性：对目标序列 检测SNP最灵敏的试剂之一 荧光背景低 优 点 只能用于一个特定目标 设计困难 价格比较高 缺 点 讲 座 提 纲 实时荧光定量PCR原理 实时荧光定量PCR的几种方法介绍 实时荧光定量PCR实验设计及应用 实时荧光定量PCR原理 实时荧光定量PCR的几种方法介绍 实时荧光定量PCR实验设计及应用 实时荧光定量PCR法 标准样品 相对定量中的内标 内标通常是β-actin、GAPDH基因等看家基因 在细胞中的表达量或在基因组中的拷贝数恒定，受环境因素影响小 内标定量结果代表了样本中所含细胞或基因组数量 绝对定量的标准样品： 已知拷贝数的质粒DNA 实时荧光定量PCR法 标准