相对定量分析PPT学习教案

1、会计学12第1页/共50页3绝对定量绝对定量典型应用领域典型应用领域: : 特定物种鉴定(e.g. bacteria, virus)特定核酸样本检测(e.g. oncology research)检测病原体载量(e.g. legionella, anthrax)筛选抗生素抗性基因(e.g. MRSA, VRE)水质监测Relative QuantificationRelative Quantification典型应用领域典型应用领域: :mRNA 表达水平的检测(e.g. 细胞因子, 肿瘤)基因定量 (e.g. 染色体缺失) 研究疾病的微小残留病灶(MRD)GMO 检测相对定量相对定量第2页/

2、共50页4目标目标需要进行研究的核酸需要进行研究的核酸 (特定的特定的RNA或或DNA序列序列)内参内参在研究的所有样本中，拷贝数恒定不变的核酸序列在研究的所有样本中，拷贝数恒定不变的核酸序列 (内源性对照内源性对照)第3页/共50页5StepStep 1:1: 相同样品中的目标基因和参比基因的浓度StepStep 2:2: 不同样品中目的基因的含量差别由参比基因校正参比基因可以修正：参比基因可以修正：样品起始量的差别样品中核酸回收率的差别样品中可能的RNA降解不同样品中核酸质量的差别加样差（目标基因浓度）（目标基因浓度）（参比基因浓度）（参比基因浓度）T = 100T = 100 T = 1

3、0 T = 10 T = 10T = 10 = 2 = 2 = 2 = 2 = 0.2= 0.2 R = 50 R = 5 R = 50 R = 5 R = 50R = 50 = = calibratorcalibratortreated cell 2treated cell 2第4页/共50页-actinmultigene family; 20 genes; 1 active locus: hormones of tyroid gland20 pseudogenes: stomach tumorg-actinmultigene family; pseudogenesGAPDHmultigen

4、e family; 10-30 genes; 200 in mouse: lung, pancreatic, colon cancermostly pseudogenes: insulin, EGF5.8S,18S, 28S RNApseudogenes2-microglobulinno pseudogenes : Non-Hodgkin lymhoma abnormal expression in tumorsG6PDHno pseudogenes: kidney, stomach tumor : hormones, oxidant stress, growth factorsPBGDno

5、pseudogenesaldolasepseudogenesHPRTpseudogenesU3, U8, .Pseudogenesornithin: tumorsdecarboxylase.GeneGenomic structure / pseudogenesRegulation e.g.第5页/共50页7第6页/共50页校准品样本： 未处理的细胞系 起始时间点的样本 正常组织样本第7页/共50页9第8页/共50页相对定量相对定量常规的常规的两种分析模式两种分析模式相对定量相对定量(以校正样本归一化处理以校正样本归一化处理)精确值精确值= 带扩增效率校正带扩增效率校正 -method 近似值近

6、似值 = 没有扩增效率校正没有扩增效率校正 CT 法法全部假设: E = 2相对定量计算公式相对定量计算公式 = 2 -CT 相对定量计算公式相对定量计算公式 =ET CpT (C) - CpT (S) X ER CpR (S) - CpR (C)校正目标基因和内参基因 实际上不同的扩增效率第9页/共50页11N = N0 x 2nNnumber of amplified molecules N0initial number of moleculesnnumber of amplification cyclesE第10页/共50页EFFICIENCY 扩增效率扩增效率ET = ER = 2 ?

7、第11页/共50页默认扩增效率默认扩增效率 E=2， (-Cp)方法方法Calibrator Normalized Ratio = 2CpT(C) CpT(S) x 2CpR(S) CpR(C) = 2CpT(C)-CpR(C) - CpT(S)-CpR(S) = 2Cp(C)- Cp(S) = 2- Cp第12页/共50页第13页/共50页实际上实际上不是所有的PCR反应都能达到E2的理想扩增效率不是所有的PCR反应在整个过程中的扩增效率都保持恒定第14页/共50页o2?n理想情况下扩增效率2Crossing Point Log Concentration样品 1 样品 2效率2 的标准曲线

8、效率偏离2的标准曲线E =10 -1/slope第15页/共50页17E = 10 -1/slope E = 10 -1/-3.703 E = 10 0.27 E = 1.86F2/F1ncp1cp2ncp3cp4cp5cp6E =10 E =10 -1/slope-1/slope第16页/共50页最为准确的相对定量结最为准确的相对定量结果果第17页/共50页校准样本校准后的浓度比校准样本校准后的浓度比 = = E ET T CpT (C) - CpT (S)CpT (C) - CpT (S) X X E ER R CpR (S) - CpR (C)CpR (S) - CpR (C) 第18页

9、/共50页20第19页/共50页21第20页/共50页22Select appropriate run protocole.g., for detection of FAM “Mono Color Hydrolysis Probe - UPL Probe” 第21页/共50页23ionnStep 2:Select SamplesnStep 3:Enter properties anddefine identifiers第22页/共50页24PropertyPropertyValid ValuesValid ValuesDescriptionDescriptionSample NameAlpha

10、numeric value (25 characters)Default value is “Sample #”Name of material of interestSample Type Unknown PositiveControl/Calibrator Negative Control Standardmandatorymandatory Type of sampleTarget Type Target Reference UnassignedmandatorymandatoryType of targetUnassigned: excluded from calculationsTa

11、rget NameAlphanumeric value (25 characters)Default value is blankName of the gene target第23页/共50页25第24页/共50页26点选 Target Name: 激活所指定的 target 点击Show Abs Quant 调整 Noise Band 点击 Calculate 点击 Back to Rel Quant第25页/共50页27第26页/共50页28第27页/共50页29第28页/共50页30第29页/共50页31选中目的基因的名字然后点击Show Abs Quant 查看并调整相关基因的扩增结

12、果 Crossing Points Results Replicate Statistics Display Efficiency 第30页/共50页32第31页/共50页33第32页/共50页34第33页/共50页35BasicBasicAdvancedAdvancedmultiple target and reference genes?YYT/R pairing rulesAll to Meanall 4calibrator allowed?YYT and R genes on different plate?NYsubset allowed?NYCp analysis methodsF

13、it pointFit point and 2nd Der. Maxstandard curve samples allowed?YYefficiency correction?YYmultiplex possible?YY第34页/共50页36第35页/共50页37相对定量相对定量 实验设计实验设计- 主要因素主要因素1. 实验设计实验设计确定研究的目的基因 选定合适的内参基因（最好能多选取几个）优化反应条件 选择合适的样本及样本制备方法(包括对照样本的选取)2. 实验验证实验验证确定实验的效果 (动态范围& 效率)3. 分析方法的选择分析方法的选择简单相对定量分析高级相对定量分析第

14、36页/共50页38第37页/共50页39(e.g. SYBR Green I) and reference (e.g. HybProbe format)n目的基因和内参基因进行多重目的基因和内参基因进行多重PCR扩增扩增 (dual color) 多重荧光扩增会降低动力学范围 比较单色扩增和双色扩增时目的基因与内参基因的动力学范围第38页/共50页40效率效率影响因子Determination of a correction factor and/or a multiplication factor单色 (目的基因和内参基因在不同的反应管或反应孔)双色 (目的基因和内参基因在同一个的反应管或

15、反应孔)flexible Pairing“ of target and reference samples (multiple housekeeping genes)反应的设置反应的设置校准样本校准样本选择合适的校准样本第39页/共50页41Established LightCycler PCR for target and reference geneDetermination of a calibratorLC run with samples and calibrator (target/reference)AnalysisBasicAdvanced第40页/共50页421.1. Exp

16、erimentExperiment DesignDesign Identification of the target gene(s) of interest and suitable reference gene(s)e.g.:target 1, target 2housekeeping gene (HK) 1, housekeeping gene (HK) 2Optimization of reaction conditionse.g.:use LightCycler 480 Probes Master together with UPL probe and primersselect t

17、he appropriate run protocol (make use of “New Experiment from Template”)Selection and preparation of sample material, including a calibrator samplee.g.:untreated sample (= calibrator)5 samples treated with different agents always include a NTCstart with cDNA prepared by Transcriptor First Strand cDN

18、A Synthesis Kit (2-step RT-PCR)Set up experimente.g.:monocolor reactiontarget and HK PCR on one MWP第41页/共50页432.2. ExperimentExperiment ValidationValidationDetermination of assay quality (dynamic range & efficiency) for each target and each reference gene3.3. MethodMethod SelectionSelection forf

19、or AnalysisAnalysisBasic Analysisfast tracking solution for well performing assaysconvenient CT MethodAdvanced Analysishigh confidence solution for most challenging assays! particular E-Method benefit of standard curves (linear, non-linear standard curve fitting; fixed dynamic range)benefit of in-ru

20、n standard (inter-assay calibration)第42页/共50页44第43页/共50页45第44页/共50页46Basic Analysisone clickFinal ResultAdvanced AnalysisFinal Resultdefine analysis settingsselect various parameters like quant type,standard curves pairing rule,reference runsubset editing adjustmentscombinable as templateadjustments

21、第45页/共50页47Basic AnalysisAdvanced Analysistarget and reference on same platefull plate analyzedAssay Set Uptarget and reference on same plateor on different plates full plate and/or subsets analyzedfixed pairing rulePairingflexible, editable pairing rulesin run or external standardsno standardsStand

22、ards(target / reference)in run or external standardsno standardsstandard curves (linear, non-linear, fixed dynamic range); E = 2, or editable efficiency valuesEfficiencystandard curves (linear, non-linear, fixed dynamic range)E = 2, or editable efficiency valuesFit Points MethodCp Analysis2nd Deriva

23、tive Max Method or Fit Pointsused & accepted approach Definitionscientifically sound approacheasy access to relative quantification data for routine applicationsCharacteristicshighest flexibility and reliability for most demanding applicationsunmatched fast tracking results(just one click!)Benefit SWunmatched flexibility fo