Introduction to Real-Time PCR & StepOne Plus TM Operation

Transcription

1 Introduction to Real-Time PCR & StepOne Plus TM Operation 林有啓 (Eugene Lin, Ph.D.) Field Application Scientist 1 The world leader in serving science

2 Normalised reporter fluorescence (R n ) Real Time PCR Amplification plot features Sample Lower expression level 0.50 lens cap PCR vessel thermal block sample Threshold Baseline region C T PCR cycle number No Template control Geometric Phase: (Exponential Phase) Y=N0(1+E) x =N0 2 n C T = threshold cycle: the calculated fractional cycle number at which the PCR product crosses a threshold of detection Rn 2

3 Real-Time PCR detection takes place in the exponential phase, while traditional detection takes place at the plateau of the reaction Plateau Linear Exponential Gel Threshold of detection fluorescent signal PCR cycles Low Ct (high copy #) High Ct (low copy #) Y= N0 2 n, CT 與起始濃度之對數值成反比 3

4 同步定量 PCR 之應用 基因定量 病毒定量 : HBV, HCV,HPV 疾病相關基因之定量 mirna 基因調控研究 sirna knock down validation 檢測基因轉殖食品 (GMO) Somatic Mutation 檢測 定性研究 基因型研究 -- SNP 與疾病關聯性 病原菌偵測 基因體拷貝數變異 (Copy Number Variants) High Resolution Melt 4

5 Generate Real-Time Signal Using Fluorescent 1. TaqMan probe or TaqMan MGB probe chemistry: 5 Nuclease assay F Q 2. SYBR Green I dye chemistry 5

6 SYBR Green Dye A minor groove -binding molecule specific to the minor groove of double-stranded DNA It fluoresces at an increased intensity when bound Major Groove Minor Groove 6

7 SYBR Green - dsdna minor-groove binding dye Polymerization Polymerization Complete 7

8 SYBR Green I Dye: Melting Curve Analysis * Use NTC to check whether non-specific product is primer dimer * If the non-specific product is primer dimer: 1. Optimize primer concentration 2. Re-design primer pair 8

9 TaqMan Chemistry- FRET Principal Excitation hv energy transfer 9

10 Fluorogenic 5' nuclease assay (TaqMan chemistry) forward primer 5' 3' R probe Q 3' 5' 5' 1. Polymerisation reverse primer 3' 5' 5' 3' 5' Q 3' 5' 3' 5' 2. Strand displacement R 5' 3' 5' 3. Cleavage Q 3' 5' 3' 5' 5' 3' R Q 5' 5' 3' 3' 5' R = Reporter Q = Quencher 4. Polymerisation completed 10

11 TaqMan MGB/NFQ Probes Minor Groove Binder (MGB) Small molecule that fits snugly into the minor groove of duplex DNA Stabilizes probe annealing Non Fluorescent Quencher (NFQ) Dark quencher Acts as energy transfer acceptor that does not emit a detectable fluorescent signal MGB probe design uses a special algorithm in Primer Express Software. All probes will be short (13-25-mers) 11

12 Real-time PCR Chemistries Specificity Sensitivity Highly specific Probe Hybridization Very High Less specific Very High Flexibility Optimization Multiplex PCR SNP detection +/- application Ready to use 20x primer/probe mix - no need to optimize Gold standard for MAQC PCR efficiency 100% ±10% No Probe is required Screening tool Need to optimize PCR program Need to check primer-dimer info Need to check PCR efficiency 12

13 2-step qrt-pcr High Capacity cdna RT Kit (P/N ) High Capacity RNA-to-cDNA (P/N ) Step1: Convert RNA to cdna (random primers/oligo dt) Multiple tubes 2hr RT Step 2: Amplify and quantify cdna (gene-specific primers) 0.5-1hr RT 13

14 Preparing Real-time PCR Reactions Reverse Transcription : High Capacity RNA-to-cDNA Kit 2X RT Buffer 20X RT Enzyme Mix Sample (up to 2μg) Nuclease-Free water 10μl 1μl Up to 9μl To 20μl Real-time PCR: TaqMan Chemistry 2x TaqMan Master Mix 1x 10μl 20x Probe/primer Assay Mix 1x 1μl Water NA cdna ng 5-10μl Standard mode PCR condition: 50, 2min 95, 10 min 95, 15 sec 40 cycles 60, 1min 20μl Fast mode PCR condition: 95, 20 sec 95, 1 sec 40 cycles 60, 20 sec SYBR Chemistry 2x Power SYBR Master Mix 1x 10μl F Primer optimized NA R Primer optimized NA Water NA cdna ng 5-10μl SYBR Green: - Check Primer Concentration: ~ nM 20μl - Add Melt (Dissociation) Curve 14

15 Real Time PCR Applications Absolute Quantitation vs. Relative Quantitation 15

16 C T values 絕對定量 (Absolute Quantitation) 主要應用於病毒量及病原菌偵測 To determine the actual number of copies of a target nucleic acid within a sample with statistical confidence. C T s C T is directly proportional to log of amount of input template Log copy number 16

17 相對定量 (Relative Quantitation) Relative n-fold difference relative to the calibrator (no units) Ct analysis (most common) relative standard curve Standard: any stock RNA or DNA containing the appropriate target Endogenous control normalized RNA input measurement and RT-efficiency difference (ex. 18S rrna, GAPDH, HPRT, -actin..) The most powerful and widely used method Ex. Time Course (Treatment) 17

18 Comparative Ct Method step 1: Normalization to endogenous control Ct Target gene Ct Endogenous control = Ct step 2: Normalization to calibrator sample Ct Sample Ct Calibrator = Ct step 3: use the formula 2 - Ct A calibrator sample is a sample to which unknown samples are compared (ex. untreated sample or control). 18

19 Comparative Ct Method Comparison of the c-myc expression level in T=0, T=12, T=24, T=48 time course study Calibrator t=0 t=12 t=24 t=48 time total RNA total RNA total RNA total RNA Spectrophotometer measure RNA quantity cdna cdna cdna cdna Reverse Transcription: Ex. 5 ug RNA/ 50 ul =100 ng/ul C-myc GAPDH C-myc GAPDH C-myc GAPDH C-myc GAPDH Ct=30.5 Ct=23.6 Ct=27 Ct=22.6 Real Time PCR Unknown samples( 50 ng): T=0, T=12, T=24, T=48 19

20 Relative Quantity of Expression ΔΔC t Calculations (Comparative C t ) c-myc GAPDH C t C t 2 - Ct T=0 (calibrator) T=12hr T=24hr T=48hr t = 0 t = 12 h t = 24 h t = 48 h 0 20

21 Applied Biosystems Primers/Probe design total solution Option 1: Pre-Designed TaqMan Assay (ready-to -use) > 1.3 million TaqMan Gene Expression assays (for 23 species) > 4.5 million TaqMan SNP assays > 1.6 million TaqMan CNV assays > 15,000 TaqMan microrna assays (mirbase release 20, 206 listed species) TaqMan Mutation Detection assays TaqMan Non-Coding RNA assays Option 2: Custom / Custom Plus Assay All-in One tube TaqMan-based Assay (20X mixture) Option 3: Primer Express software Software for designing real-time assays 上機條件皆相同 ~~ 不用再花時間測試 primer 溫度了 21

22 How to search AB TaqMan assay on line? Gene name or RefSeq Accession. 22

23 TaqMan Gene Expression Array Plates 23

24 Pre-configured TaqMan Gene Expression Array Plate Pre-configured plates for 135 different diseases, pathways, and biological processes 96-well Fast(0.1 ml) plate Species Human Mouse Rat Human Alzheimer's Disease Human Angiogenesis Human Apoptosis Human Immune Response Human Inflammation Human Phosphodiesterase Human Protein Kinase Pathways Human Stem Cell Pluripotency Mouse Alzheimer's Disease Mouse Immune Response Mouse Stem Cell Pluripotency Rat Inflammation Rat Phosphodiesterase Human Androgens Human Chemokines Human CYP450 and other Oxygenases Human Diabetes Human Estrogens Human Heat Shock Proteins Human Hedgehog Pathway Human Hypoxia Human LDL Pathways Human Ligand Gated Ion Channels Human MAP Kinase Pathways Human Neurotransmitters Human Notch Signaling Human ABC Transporters Human p53 Signaling Human CMV & MAPK Pathways Human PDGF Pathway Human Cholera Infection Human Phagocytosis of Microbes Human Pathogenesis of ALS Human ABC Transporters Human p53 Signaling Human CMV & MAPK Pathways Human PDGF Pathway Human Cholera Infection Human Phagocytosis of Microbes Human Pathogenesis of ALS Human TNF Superfamily Pathway Human TNFR1 Pathway Human TNFR2 Pathway Human Toll Comparative Pathway Human Toll-Like Receptors Pathway Human Transcription of mrna Human Transcription of rrna Human Transcription of trna Mouse lipid regulated genes Rat Endogenous Controls Mouse Endogenous Controls Human Androgens Human Chemokines Human Diabetes Human Estrogens Human Growth Factors Human Hematopoisis Human NGF Pathway Human NFkB Pathway Human Osteogenesis Human TGFB Pathway Human Tumor Metastasis Human TNFR1 Pathway Human TNFR2 Pathway Human WNT Pathway Human Hox Genes Human BMP Pathway Human IL-2 Pathway Human IL-9 Pathway Human inos Signaling Human IL-1 Pathway Human IL-10 Pathway Human Growth Factors Human Hematopoisis Human EGF Pathway Human FGF Pathway 24

25 Targets and Pathway Information 25

26 Plate Layout and Assay ID Assay ID A Hs _s1 Hs _m1 Hs _m1 Hs _m1 Hs _m1 Hs _m1 Hs _m1 Hs _m1 Hs _m1 B Hs _m1 Hs _m1 Hs _m1 Hs _m1 Hs _m1 Hs _m1 Hs _m1 Hs _m1 Hs _s1 C Hs _m1 Hs _m1 Hs _m1 Hs _m1 Hs _m1 Hs _m1 Hs _m1 Hs _m1 Hs _m1 D Hs _m1 Hs _m1 Hs _m1 Hs _m1 Hs _m1 Hs _m1 Hs _m1 Hs _m1 Hs _m1 E Hs _m1 Hs _m1 Hs _m1 Hs _m1 Hs _m1 Hs _m1 Hs _m1 Hs _m1 Hs _m1 F Hs _gH Hs _m1 Hs _m1 Hs _m1 Hs _m1 Hs _s1 Hs _m1 Hs _m1 Hs _m1 G Hs _m1 Hs _m1 Hs _m1 Hs _s1 Hs _m1 Hs _m1 Hs _m1 Hs _m1 Hs _m1 H Hs _m1 Hs _s1 Hs _m1 Hs _m1 Hs _m1 Hs _m1 Hs _g1 Hs _m1 Hs _m1 Gene Symbol A 18S GAPDH HPRT1 GUSB ACVR1 ACVR1B ACVR2A ACVR2B AMH B BMP3 BMP4 BMP5 BMP6 BMP7 BMPR1B BMPR2 CREBBP DCN C FMOD FNTA GDF2 MSTN GDF9 GDF10 IFNG IL6 INHA D LTBP1 LTBP2 LTBP3 SMAD1 SMAD2 SMAD3 SMAD4 SMAD5 SMAD6 E PPP2CA PPP2CB MAPK1 MAPK3 RBL1 RBL2 ROCK1 BMPER TSC22D1 F TFDP1 TGFA TGFB1 TGFB2 TGFB3 LEFTY2 TGFBR1 TGFBR2 TGFBR3 G CUL1 CHRD BMP15 NOG ZFYVE9 TGFBRAP1 ROCK2 GDF15 GDF3 H FST LEFTY1 BMP10 HIPK2 SMURF1 SMURF2 INHBE IL17F ACVR1C 26

27 Configure Custom TaqMan Array Plate TaqMan Array Plates Fixed and custom formats: 8, 16, 32, 48, and well plates Uses inventoried assays 10µl reaction volume for StepOnePlus 27

28 What are SNPs? 單核苷酸多型性 (Single Nucleotide Polymorphism, 簡稱 SNP) 指的是 DNA 序列上發生的單個核苷酸鹼基之間的變異 人類遺傳基因的各種差異, 有 90% 都可歸因於 SNP 的基因差異 診斷及預測致病風險評估 藥物基因體學及新藥的開發 Mom Dad Each person has 2 copies or 2 alleles of each gene 1 allele on each chromosome. Each person receives 1 allele from each parent. If both alleles are the same, the person is homozygous for that gene. If the alleles differ, then the person is heterozygous for that gene. 28

29 TaqMan SNP Genotyping Assay Overview 29

30 Allelic Discrimination (SNP) Data A Homozygous AA Heterozygous GA Homozygous GG G 30

31 mirna Quantitation by Real-Time PCR Mature mirna Northern Blot Result Assay for let - 7a Synthetic mirna Target let - 7a let - 7b let - 7c let - 7d 31

32 Individual TaqMan MicroRNA Assays Aligned with release 19 of the mirbase > 12,000 TaqMan microrna assays Assay include: Human, Mouse, Rat, Drosophila melanogaster, Caenorhabditis elegans, Arabidopsis thaliana 193 listed species 46 endogenous controls (small nuclear RNAs) U6 snrna, RNU6B, RNU43, RNU44, RNU48, Z30, snorna202.. Each assay contain: 1 RT primer, 1 TaqMan Assay Additional Applied Biosystems Products required to run mirna assays -- TaqMan MicroRNA RT kit (200 rxn, / or 1000 rxn, ) -- TaqMan Universal PCR Master Mix 32

33 Copy Number Variation (CNV)- 基因拷貝數變異 Copy Number Variation (CNV) A structural genomic variant involving copy number changes in comparison to a reference genome Deletion or duplication events involving>1 kb of DNA. Most are <10 Kb; some rare CNVs >1 Mb CNVs are found in normal individuals and have also been associated with disease and other phenotypes 異常的 DNA 拷貝數變化 (CNV) 是許多疾病 ( 如癌症 遺傳性疾病 心血管疾病 ) 的一種重要分子機制. 傳統的方法 ( 比如 G 顯帶,FISH, CGH 等 ) 存在操作繁瑣, 解析度低等問題, 難以提供變異區段的具體資訊 33

34 Workflow of TaqMan Copy Number Variation Assays > 1.6M Pre-Designed TaqMan Copy Number Assays available FAM -labeled TEST ASSAY 1 CopyCaller VIC -labeled CONTROL ASSAY (i.e.: RNase P) 2 Standard TaqMan protocol gdna 1 ng / µl PCR rxn 3 qpcr TaqMan Master Mix 4 4 replicates per gdna sample 34

35 Determination of DNA Copy Number 35

36 CopyCaller Software- 輕鬆獲得 CNV 結果 Flexible 不需要已知拷貝數的樣品當 control Free 免費下載分析軟體 Easy to use 幾分鐘內完成分析, 搭配圖形化介面, 輕鬆了解判讀結果 Results with confidence value 軟體內建統計運算邏輯, 提供值得信賴的結果 36

37 TaqMan Mutation Detection Assays (TMDA) Somatic Mutation Detection by castpcr Technology TMDA Product Line Summary Assays for 778 key mutations from 46 cancer genes Corresponding gene reference assays Wild-type assays for a subset of mutation targets Internal Positive Control Reagents (IPC kit) Mutation Detector Software Somatic mutations reported in the important genes related to biological pathways such as EGFR, Ras-Raf, KIT, FLT3, and PDGFRA High sensitivity (0.1-1%) for use with FFPE samples and biopsies High specificity for generating accurate results Gene List AKT1 ALK APC BRAF CDKN2A CTNNB1 EGFR FGFR3 FLT3 GNAS HRAS IDH1 JAK2 KRAS KIT MPL NPM1 NRAS PDGFRA PIK3CA PTEN TP53 VHL 37

38 TaqMan Mutation Detection Assays (TMDA) Superior Sensitivity 0.1 % High Specificity Simple and scalable workflow 3 hrs from sample to results Competitive Allele-Specific TaqMan PCR - castpcr 38

39 Mutation Detector Software 39

40 定量 PCR Primers/ Probe 設計軟體 40

41 3. 定量 PCR Primers/ Probe 設計軟體 清楚明確的 TaqMan Probe & Primer 設計規範 200 bp amplicon 500 bp amplicon 41

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43 2. Find Primer/Probe 1. Add DNA file or Copy & Paste 43

44 Design Parameter 44

45 Result 45

46 Check Tm of Primers 46

47 Ct value SYBR Green experiment Note 1. Primer conc. Optimization Primer Final conc nm No primer dimer or non-specific product involved 2. PCR Primer Efficiency Validation Sample serial dilution to run standard curve for target gene and endogenous control gene 3. Real sample run for each gene Ct C-Myc Log of Input GAPDH 47

48 The StepOnePlus Real-Time PCR System 簡易三步驟!! 48

49 StepOnePlus Real-Time PCR System: The Basics 4-color instrument FAM /SYBR Green dyes VIC /JOE dyes NED /TAMRA dye ROX dye 49

50 StepOnePlus Real-Time PCR System: The Basics 96-Well Block - One block, 2 speeds Fast cycling: 40 cycles in under 40 minutes Standard cycling: 40 cycles in under 2 hours 10-30µl reaction volume 4 50

51 樣品量多時 StepOnePlus Compatible Consumables P/N MicroAmp Fast 96-Well Reaction Plate (0.1 ml) -10 plates P/N MicroAmp Optical Adhesive Film - 25 films 樣品量少時 P/N MicroAmp Fast 8-Tube Strip (0.1 ml) strips P/N MicroAmp Optical 8-Cap Strip strips Place the tray containing the tube, Load at least 16 tube 51

52 The flat edge of an applicator is rubbed back-and-forth along the length of the plate with a significant downward pressure to form a complete seal on top the wells Downward pressure applied in back-and-forth motions across the top of the plate Note: Pressure is required to activate the adhesive on the optical cover 52

53 Operation Notes Directly load fast optical 96-well plate into the instrument If using fast 8-tube stripes, load the tubes with fast 96- well tray Do not mark any labels on the consumables This may increase the background signal Avoid bubbles when pipetting into each well Centrifuge samples 53

54 Standalone (PC-Free) 只需輕按觸控螢幕不需電腦連線也能上機!! 1. Start the run from the touchscreen 2. After run, download the file (.eds) to your PC 3. Analyze your data 54

55 Browse/New Experiments: New 55

56 Select Experiment : Save 56

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59 Standalone: 只能暫存一個檔案 插入 USB, 待 icon 出現在右下角, 即可點選 Collect Results 檔案會自動存到 USB 中 59

60 StepOne TM v2.3 Software 一套軟體可以符合全方位的應用 (1280x1024 pixel resoltion) 絕對定量 Quantification - Standard Curve 相對定量 Quantification Comparative Ct ( Ct) 相對定量 Quantification - Relative Standard Curve Melting Curve Analysis Genotyping Presence/Absence 60

61 1. Run: QuickStart 61

62 2. Setup: Experiment Properties a. Experiment Name 及檔案儲存位置 b. 選擇 Experiment Type c. 選擇使用螢光系統 d. 選擇 Ramp Speed e. 選擇實驗樣品種類 62

63 3. Setup: Run Method 4. 63

64 5. Setup: Plate Setup 定義基因和樣品名稱 輸入偵測的基因及使用的螢光 輸入樣品名稱 64

65 6. Setup: Plate Setup 決定基因和樣品位置 圈選樣品擺放位置, 再從左邊勾選樣品名稱與偵測的基因 選擇 Reference Sample & Endogenous Control Gene 65

66 6. Setup: Plate Setup 決定標準品位置 Automatic Standard Curve Setup 圈選樣品擺放位置, 再從左邊勾選樣品名稱與偵測的基因 66

67 6. Setup: Plate Setup 決定標準品位置 Automatic Standard Curve Setup 67

68 7. Analysis: Amplification Plot 3. Analyze or Re-analyze Auto or Manual 4. Check Threshold 68

69 Analysis: View Well Table 69

70 Analysis: Gene Expression 70

71 Analysis: Standard Curve 71

72 Analysis: Melting Curve (SYBR Green) 72

73 Analysis: Multicomponent Plot 73

74 Analysis: QC Summary Helps with Troubleshooting 74

75 數據和圖形簡易輸出! 超 easy~ Export to Excel, PowerPoint or save as jpeg 75

76 StepOnePlus Real-Time PCR System: The Basics Veriflex Block -One block, Six Zones -The same Better than gradient feature from Veriti 96-well Thermal Cycler *Image from Veriti Thermal Cycler 76

77 Red lines to delineate zones 77

78 Setup: Run Method 78

79 Useful information-- 中文線上課程 79

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