相對定量分析PPT學(xué)習(xí)教案

1、會計學(xué)12第1頁/共50頁3絕對定量絕對定量典型應(yīng)用領(lǐng)域典型應(yīng)用領(lǐng)域: : 特定物種鑒定(e.g. bacteria, virus)特定核酸樣本檢測(e.g. oncology research)檢測病原體載量(e.g. legionella, anthrax)篩選抗生素抗性基因(e.g. MRSA, VRE)水質(zhì)監(jiān)測Relative QuantificationRelative Quantification典型應(yīng)用領(lǐng)域典型應(yīng)用領(lǐng)域: :mRNA 表達(dá)水平的檢測(e.g. 細(xì)胞因子, 腫瘤)基因定量 (e.g. 染色體缺失) 研究疾病的微小殘留病灶(MRD)GMO 檢測相對定量相對定量第2頁/

2、共50頁4目標(biāo)目標(biāo)需要進(jìn)行研究的核酸需要進(jìn)行研究的核酸 (特定的特定的RNA或或DNA序列序列)內(nèi)參內(nèi)參在研究的所有樣本中，拷貝數(shù)恒定不變的核酸序列在研究的所有樣本中，拷貝數(shù)恒定不變的核酸序列 (內(nèi)源性對照內(nèi)源性對照)第3頁/共50頁5StepStep 1:1: 相同樣品中的目標(biāo)基因和參比基因的濃度StepStep 2:2: 不同樣品中目的基因的含量差別由參比基因校正參比基因可以修正：參比基因可以修正：樣品起始量的差別樣品中核酸回收率的差別樣品中可能的RNA降解不同樣品中核酸質(zhì)量的差別加樣差（目標(biāo)基因濃度）（目標(biāo)基因濃度）（參比基因濃度）（參比基因濃度）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頁/共50頁-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頁/共50頁7第6頁/共50頁校準(zhǔn)品樣本： 未處理的細(xì)胞系 起始時間點(diǎn)的樣本 正常組織樣本第7頁/共50頁9第8頁/共50頁相對定量相對定量常規(guī)的常規(guī)的兩種分析模式兩種分析模式相對定量相對定量(以校正樣本歸一化處理以校正樣本歸一化處理)精確值精確值= 帶擴(kuò)增效率校正帶擴(kuò)增效率校正 -method 近似值近

6、似值 = 沒有擴(kuò)增效率校正沒有擴(kuò)增效率校正 CT 法法全部假設(shè): E = 2相對定量計算公式相對定量計算公式 = 2 -CT 相對定量計算公式相對定量計算公式 =ET CpT (C) - CpT (S) X ER CpR (S) - CpR (C)校正目標(biāo)基因和內(nèi)參基因 實際上不同的擴(kuò)增效率第9頁/共50頁11N = N0 x 2nNnumber of amplified molecules N0initial number of moleculesnnumber of amplification cyclesE第10頁/共50頁EFFICIENCY 擴(kuò)增效率擴(kuò)增效率ET = ER = 2 ?

7、第11頁/共50頁默認(rèn)擴(kuò)增效率默認(rèn)擴(kuò)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頁/共50頁第13頁/共50頁實際上實際上不是所有的PCR反應(yīng)都能達(dá)到E2的理想擴(kuò)增效率不是所有的PCR反應(yīng)在整個過程中的擴(kuò)增效率都保持恒定第14頁/共50頁o2?n理想情況下擴(kuò)增效率2Crossing Point Log Concentration樣品 1 樣品 2效率2 的標(biāo)準(zhǔn)曲線

8、效率偏離2的標(biāo)準(zhǔn)曲線E =10 -1/slope第15頁/共50頁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頁/共50頁最為準(zhǔn)確的相對定量結(jié)最為準(zhǔn)確的相對定量結(jié)果果第17頁/共50頁校準(zhǔn)樣本校準(zhǔn)后的濃度比校準(zhǔn)樣本校準(zhǔn)后的濃度比 = = 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頁

9、/共50頁20第19頁/共50頁21第20頁/共50頁22Select appropriate run protocole.g., for detection of FAM “Mono Color Hydrolysis Probe - UPL Probe” 第21頁/共50頁23ionnStep 2:Select SamplesnStep 3:Enter properties anddefine identifiers第22頁/共50頁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頁/共50頁25第24頁/共50頁26點(diǎn)選 Target Name: 激活所指定的 target 點(diǎn)擊Show Abs Quant 調(diào)整 Noise Band 點(diǎn)擊 Calculate 點(diǎn)擊 Back to Rel Quant第25頁/共50頁27第26頁/共50頁28第27頁/共50頁29第28頁/共50頁30第29頁/共50頁31選中目的基因的名字然后點(diǎn)擊Show Abs Quant 查看并調(diào)整相關(guān)基因的擴(kuò)增結(jié)

12、果 Crossing Points Results Replicate Statistics Display Efficiency 第30頁/共50頁32第31頁/共50頁33第32頁/共50頁34第33頁/共50頁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頁/共50頁36第35頁/共50頁37相對定量相對定量 實驗設(shè)計實驗設(shè)計- 主要因素主要因素1. 實驗設(shè)計實驗設(shè)計確定研究的目的基因 選定合適的內(nèi)參基因（最好能多選取幾個）優(yōu)化反應(yīng)條件 選擇合適的樣本及樣本制備方法(包括對照樣本的選取)2. 實驗驗證實驗驗證確定實驗的效果 (動態(tài)范圍& 效率)3. 分析方法的選擇分析方法的選擇簡單相對定量分析高級相對定量分析第

14、36頁/共50頁38第37頁/共50頁39(e.g. SYBR Green I) and reference (e.g. HybProbe format)n目的基因和內(nèi)參基因進(jìn)行多重目的基因和內(nèi)參基因進(jìn)行多重PCR擴(kuò)增擴(kuò)增 (dual color) 多重?zé)晒鈹U(kuò)增會降低動力學(xué)范圍 比較單色擴(kuò)增和雙色擴(kuò)增時目的基因與內(nèi)參基因的動力學(xué)范圍第38頁/共50頁40效率效率影響因子Determination of a correction factor and/or a multiplication factor單色 (目的基因和內(nèi)參基因在不同的反應(yīng)管或反應(yīng)孔)雙色 (目的基因和內(nèi)參基因在同一個的反應(yīng)管或

15、反應(yīng)孔)flexible Pairing“ of target and reference samples (multiple housekeeping genes)反應(yīng)的設(shè)置反應(yīng)的設(shè)置校準(zhǔn)樣本校準(zhǔn)樣本選擇合適的校準(zhǔn)樣本第39頁/共50頁41Established LightCycler PCR for target and reference geneDetermination of a calibratorLC run with samples and calibrator (target/reference)AnalysisBasicAdvanced第40頁/共50頁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頁/共50頁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頁/共50頁44第43頁/共50頁45第44頁/共50頁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頁/共50頁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