The AB7900Fast and Principles of Real Time PCR

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1 The AB7900Fast and Principles of Real Time PCR Michael Tavaria, PhD Scientific Applications Specialist, Applied Biosystems, Australia and New Zealand

2 Outline Chemistries 7900Fast Real Time PCR System Applications Reaction Efficiency Reverse Transcription Singleplex/Multiplex Assay Options

3 Real-Time PCR Detection What is it? - the automatic detection of PCR product growth throughout the amplification process. Collect geometric phase data. Rn No post-pcr steps. threshold Enables high precision and high throughput. cycles

4 Outline Chemistries 7900Fast Real Time PCR System Applications Reaction Efficiency Reverse Transcription Singleplex/Multiplex Assay Options

5 Real Time PCR Chemistry SYBR green I dye (binds to ds DNA) 5 nuclease assay (TaqMan probe)

6 SyBr Green 1 Generic dye that binds to all amplified DNA including non-specific products and primer dimers Useful for screening large numbers of genes eg. dozens Limited precision, especially low copy number Primer design critical requires time, reagents, samples etc testing and validation

7 TaqMan (5 nuclease) Assay TaqMan probe R 5 Fluorescent Reporter Dye Q 3 Quencher Intact probe does not fluoresce (FRET) During amplification process, probe is cleaved by 5 - nuclease activity of Taq polymerase

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

9 TaqMan (5 nuclease) Assay Denaturation

10 TaqMan (5 nuclease) Assay Denaturation

11 TaqMan (5 nuclease) Assay Denaturation

12 TaqMan (5 nuclease) Assay Denaturation

13 TaqMan (5 nuclease) Assay Annealing of Primers and Probe

14 TaqMan (5 nuclease) Assay Annealing of Primers and Probe

15 TaqMan (5 nuclease) Assay Annealing of Primers and Probe

16 TaqMan (5 nuclease) Assay Polymerization and Probe Cleavage

17 TaqMan (5 nuclease) Assay Polymerization and Probe Cleavage

18 TaqMan (5 nuclease) Assay Polymerization and Probe Cleavage

19 TaqMan (5 nuclease) Assay Polymerization and Probe Cleavage

20 TaqMan (5 nuclease) Assay Polymerization and Probe Cleavage R

21 TaqMan (5 nuclease) Assay Polymerization and Probe Cleavage R

22 TaqMan (5 nuclease) Assay Polymerization and Probe Cleavage R

23 TaqMan (5 nuclease) Assay Polymerization and Probe Cleavage R

24 TaqMan Probes 1. Conventional 3 TAMRA fluorescent quencher Average length for 69C Tm = 24ntd R Q 2. MGB Minor Groove Binder Non-fluorescent 3 quencher Average length for 69C Tm = 16ntd R NFQ MGB

25 TaqMan (5 nuclease) Assay The TaqMan probe provides: Increased specificity Lower background noise than SyBr Highest sensitivity Greater accuracy/precision More reproducible

26 Outline Chemistries 7900Fast Real Time PCR System Applications Reaction Efficiency Reverse Transcription Singleplex/Multiplex Assay Options

27 AB 7900HT Fast Real Time PCR System The Gold Standard in Real Time PCR

28 AB 7900HT Fast Real Time PCR System System Features: Four interchangeable block formats Optional Automation Accessory & Barcode Scanner Argon ion laser/ccd camera Easy to Use Software, Multiple Applications Set up Wizards QC Filtering/Flag System Flexible data reports & exporting

29 AB 7900HT Fast Real Time PCR System Laser CCD Lens Grating Emmision filter Beam splitter Advanced Optics Argon Ion Laser 500nm 660nm range Sequential 5nm data capture Multicomponent dye analysis >10,000 hour lifespan FAM VIC NED ROX Fresnel lens Side view 384 well plate Front view

30 AB 7900HT Fast Real Time PCR System Unmatched Flexibility and Performance: Four Interchangeable Blocks 96 well Standard or Fast 384 well TaqMan Array Quickly and easily change blocks 5 mins hands on No recalibration required Excellent Block Uniformity Roche recommend AB9700 blocks for diagnostic PCR testing

31 AB 7900HT Fast Real Time PCR System Unmatched Flexibility and Performance: TaqMan MicroFluidic Card Array 384 well pre-dispensed wells Pre-designed panels or custom 1-8 samples per array assays per array Pre-designed or Custom TaqMan Array Plates 96 or 384 well pre-dispensed assays Multiple configurations Individual Assays

32 AB 7900HT Fast TaqMan MFC Arrays Multiple custom 384 well formats Ideal for analysing panels of genes Choose from over 40,000 assays TaqMan Gene Expression Assays covering human, mouse, and rat genes. Easy to load and run Load Spin Seal Run time 5 minutes!

33 AB 7900HT Fast TaqMan Arrays 1 to to 8 Samples 384 wells 1ul/well to to 380 Targets (per (per one one card) card) 1, 1, 2 or or 4 Replicates

34 AB 7900HT Fast TaqMan Arrays Pre-mix cdna & MasterMix Pipette into loading ports Centrifuge Card Sealing Analyse Results Perform Real Time PCR Remove Loading Ports

35 AB 7900HT Fast TaqMan Arrays Unmatched Flexibility and Performance:

36 AB 7900HT Fast Real Time PCR System Minimum Supported Sample Volumes: 96 well Std 20ul for quantitation 10ul for genotyping 96 well Fast 10ul for all applications 384 well 10ul for quantitation 5ul for genotyping TLDA 2ul/well for filling 1ul/well actual volume

37 AB 7900HT Fast Real Time PCR System Run Options: 96 well Std Block Must use Std 96 well Optical Plates or Tubes 7900 Std Run + Universal Master Mix approx 90mins/run 96 well Fast Block Must use 96 well Optical Fast Plates or Tubes 7900 Fast Run + Fast Master Mix approx 40mins/run 7900 Std Run + Universal Master Mix approx 90mins/run 384 well (Not a Fast Block) Use Std 384 Optical Plates 7900 Std Run + Universal Master Mix approx 90mins/run 7900 semi fast Run + Fast Master Mix approx 50mins/run (95C 20 secs, 40x (95C 1 sec, 60C 20 secs))

38 Easy Reaction Setup 2x PCR Mix + = AmpliTaq Gold DNA Polymerase Reaction buffer dntps with dutp Uracil N Glycosylase (UNG) Passive reference dye - ROX Primers and Probe PCR Premix sample DNA

39 Universal Thermal Cycling Conditions Time approx. 1:30 hr UNG -Taq Activ n Thermal Cycling

40 Easy FAST Reaction Setup 96 well FAST plates 2x FAST PCR Mix + = FAST Taq DNA Polymerase Reaction buffer dntps with dutp Passive reference dye - ROX Primers and Probe PCR Premix sample DNA

41 Universal FAST Thermal Cycling Conditions Time approx. 50 mins -Taq Activ n Thermal Cycling

42 Outline Chemistries 7900Fast Real Time PCR System Applications Reaction Efficiency Reverse Transcription Singleplex/Multiplex Assay Options

43 AB 7900HT Fast Real Time PCR System Broad Range of Applications: Absolute Quantitation (AQ) Relative Quantitation (RQ) Allelic Discrimination (SNP Genotyping) Plus/Minus High Resolution Melting (HRM) Protein Melting

44 AB 7900HT Fast Real Time PCR System Applications Quantitation: Absolute Quantitation using Standard Curve Relative Quantitation Standard Curve Comparative ( Ct) Powerful RQ Study Multi-plate Studies Manual or Automatic Analysis Settings Flexible data export functions

45 AB 7900HT Fast Real Time PCR System Applications - Allelic Discrimination: TaqMan SNP Genotyping Assays Over 1 million pre-designed and guaranteed human and mouse SNP assays Universal conditions and reagents Powerful clustering algorithm Manual or Automatic Analysis Settings

46 AB 7900HT Fast Real Time PCR System Applications - Plus/Minus Assays: Custom or pre-designed assays Internal PCR Control (IPC) Reagent Monitors for PCR inhibitors Prevents reporting of False Negatives Manual or Automatic Analysis Settings Flexible data export functions

47 AB 7900HT Fast Real Time PCR System Applications - HRM High Resolution Melt curve analysis Uses Saturating Dyes such as Syto9, LC Green, Eva Green Fluorescent normalisation required to group samples based on curve shape High number of data capture points Multiple applications

48 AB 7900HT Fast Real Time PCR System HRM - Multiple Applications: SNP Genotyping Mutation Screening % Methylation Viral/Microbial Identification Many other applications

49 AB 7900HT Fast Real Time PCR System AB HRM Software Powerful and Simple: Manual or Automatic Setting of Normalisation Regions Minimal subjective analysis Variants Clustered without need for Controls Multiple data views Raw, Normalised, Difference Plots Multiple targets per plate Flexible data display options

50 AB 7900HT Fast Real Time PCR System HRM Software Summary: Easy to Use Optimised default pre- and post-melt regions OR Use derivative melt view to manually set Powerful & Accurate clustering algorithm Automatic Calling of variant groups without need for controls Ability to analyse multiple assays Display plots coloured by sample or genotype Easy to assign genotype controls Easy to manually assign genotypes Flexible export options

51 Real Time PCR Absolute Quantification Aim: To describe the amount of a target nucleic acid in a sample with accurate units. Common Uses: Pathogen quantification Quality control Forensics mrna expression? Requires: Standard of known concentration.

52 Real Time PCR Relative Quantification Aim: To describe the concentration of a target nucleic acid in a sample relative to a calibrator/reference sample. Common Uses: mrna/mirna expression Methods: (i) Relative standard curve (ii) Comparative Ct ( Ct) Requires endogenous control

53 Relative Quantification (i) - Standard Curve Target/sample diluted to relative std curve Little validation and optimisation Immediate results Different reaction efficiencies OK Requires accurate pipetting and/or multiple replicates

54 Relative Quantification Ct Method No standard curves Reaction efficiencies must be similar (+/- 10%) High throughput Requires validation

55 Relative Quantification Ct) ( The Ct: Compare Ct value of target gene (A) to Ct value of endogenous control housekeeping gene (B) Threshhold Ct

56 Relative Quantification Ct) ( In a 100% efficient PCR reaction, each cycle represents a doubling of product 2 Ct = relative expression level eg. Ct (end. control) = 26 Ct (target) = 29 Relative expression = 2 Ct = 2 3 = 8 Ct = 3 ie. There is 8 times as much endog. control gene as target gene in that sample

57 Relative Quantification Ct) ( The Ct: A study will usually involve comparing two different sample types eg. Normal vs Tumour Now, it is the Ct that is important ie. Ct = Ct (Tumour) - Ct (Normal) Therefore 2 Ct = relative expression level

58 Relative Quantification Ct) ( If Ct (Normal) = 3, Ct (Tumour) = 8 Relative expression = 2 Ct = = 2-5 = 1/32 ie. There is 32 times as much target gene in Normal compared to Tumour Expression of Gene X in Normal vs Tumour 40 Relative Expression Gene X could be a tumour suppressor! 0 Normal Tumour

59 RQ - Endogenous Controls All relative quantitation ( Ct and Std curve) requires an endogenous control or housekeeping gene to normalise for: 1. the amount of (RNA in the) sample 2. the amount of cdna made 3. pipetting errors

60 RQ - Endogenous Controls The endogenous control gene should be: (i) present in all samples and (ii) expressed at a constant level Commonly used EC genes include: 18S rrna GAPDH B actin PGK B2M etc

61 RQ - Endogenous Controls Choosing an endogenous control: You MUST Choose Wisely Literature search Determine experimentally 96 well Endogenous Control Plate Important to verify constant expression in your experimental system

62 Outline Chemistries 7900Fast Real Time PCR System Applications Reaction Efficiency Reverse Transcription Singleplex/Multiplex Assay Options

63 Relative Quantification Ct) ( Reaction Efficiency Considerations For accurate relative quantitation using the Ct analysis method, it is important that the efficiency of the endogenous control and target reaction(s) are similar. Otherwise, the relative standard curve method should be used or the reactions should be re-designed.

64 Relative Quantification Ct) ( Reaction Efficiency Considerations A 100% efficient reaction will amplify a target 10-fold in 3.32 cycles (ie = 10 = 100%) Eff = [10 (-1/slope) 1] x 100 Reaction efficiencies of target and endogenous control should be +/- 10% of each other

65 Relative Quantification Ct) ( Reaction Efficiency Considerations Calculate reaction efficiency from the slope of the std curve:

66 Relative Quantification Ct) ( Reaction Efficiency Considerations Important Considerations Well calibrated pipettes Good pipetting technique Consistent preparation Consistent reagents Consistent instrument Measure over large dynamic range Sample purity/inhibitors

67 Outline Chemistries 7900Fast Real Time PCR System Applications Reaction Efficiency Reverse Transcription Singleplex/Multiplex Assay Options

68 Reverse Transcription cdna synthesis priming: Oligo(dT) vs Random Primer vs Gene Specific 1-step vs 2-step RT Reagent Kits (all RNaseH +ve): High Capacity cdna Reverse Transcription Kit High Capacity RNA-to-cDNA Master Mix or Kit TaqMan or SyBr Green RNA-to-Ct 1-step or 2-step Kits Taqman Gene Expression Cells-to-Ct Kits

69 Solutions for limited sample/low abundance targets 1. TaqMan PreAmp Master Mix Kit Multiplex cdna pre-amplification of up to 100 gene targets Does not introduce amplification bias preserves target stoichiometry Sample-stretcher Get more data from limited samples, such as biopsies, LCM, tumor tissue, and single cells Works on randomly primed cdna, not 3 -biased Designed to work with TaqMan Gene Expression Assays and TaqMan Universal PCR Master Mix Short amplicons for TaqMan Assays enable preamplification of FFPE samples

70 How does PreAmp work? mrna Extraction Pre Amplification cdna synthesis -High Capacity cdna Reverse Transcription Kit Real Time PCR

71 How does PreAmp work? ng of RNA is converted to cdna using the High Capacity cdna archive kit. Preamplification cocktail is created by pooling up to 100 TaqMan Gene Expression Assays (primers and probe) and mixing with cdna and TaqMan PreAmp Master Mix Preamplification reaction is thermal cycled for 10 or 14 cycles Preamplification reaction is then diluted and dispensed into single-plex TaqMan real-time PCR reactions

72 How does PreAmp work? Pool up to 100 TaqMan Gene Expression Assays Prepare RNA and Reverse Transcribe Pooled TaqMan Assays + cdna + TaqMan PreAmp Master Mix (2x) Perform PreAmplification PCR (10 14 Cycles) Dilute PreAmplification Product Diluted PreAmplification Product TaqMan + Universal PCR + Master Mix (2x) TaqMan Gene Expression Assay Perform Real-Time PCR Amplification and Analyze Data

73 Solutions for limited sample/low abundance targets What is it? 2. TaqMan Cells to Ct Kit A complete solution from Sample Prep to Real-Time PCR from cultured cells. Eliminates the need for traditional RNA purification The kit includes all the reagents for each step of the workflow: lysis (including DNase treatment), Reverse Transcription and PCR using the new TaqMan Gene Expression Master Mix Advantages Reduced labor time Easy to use, validated workflow High throughput applications Limited sample

74 TaqMan Cells to Ct Kits Procedure 3 Steps: 1. Lysis at Room Temp 2. Reverse Transcription 3. Real-time PCR Up to 45% of the RT reaction can be made up of lysate

75

76 Outline Chemistries 7900Fast Real Time PCR System Applications Reaction Efficiency Reverse Transcription Singleplex/Multiplex Assay Options

77 Singleplex or Multiplex? Singleplex Multiplex - immediate results - no validation - requires accurate pipetting - more cost effective for large number of target genes - requires validation - reduces need for accurate pipetting - more cost effective for small number of target genes and/or long term studies

78 Dye Choices when Multiplexing Multiplex level Instrument HT 1 st Reporter FAM FAM FAM FAM 2 nd Reporter VIC VIC VIC VIC 3 rd Reporter NED / NED / NED / NED TAMRA TAMRA TAMRA 4 th Reporter Cy5 Passive Reference ROX ROX ROX ROX Things to avoid FAM/TET duplex FAM/TET duplex FAM/TET duplex

79 Outline Chemistries 7900Fast Real Time PCR System Applications Reaction Efficiency Reverse Transcription Singleplex/Multiplex Assay Options

80 Assay Design Options 1. Validated TaqMan GeneEx/SNP Assays Guaranteed pre-optimised assays to most genes in many useful species eg. human, mouse, rat, drosophila 2. Custom TaqMan GeneEx/SNP Assays Submit your own sequence 3. TaqMan Express Plating Service Pre-plated assays in multiple formats 4. Primer Express TM (SyBr, TaqMan ) Design your own assays

81 Custom Primer Design Software

82 Primer Express Design Guidelines Primers Probes %GC in the range 30 80% No runs of more than 3 consecutive Gs Primer Express Tm of 58-60ºC Primer Express Tm of 68-70ºC No more than 2 Gs and Cs in last 5 bases at 3 end No G in first 2 bases at 5 end No more than 5 consecutive As Avoid GGG or GGAG at 3 end Recommended amplicon size No more than 1 CC near middle Select strand with more Cs than Gs bp

83 Suggested Reading Real Time PCR Chemistry Guide White Paper Amplification Efficiency of TaqMan Assays-on-Demand Gene Expression Products White Paper - 5 Nuclease Assays for Validating Hits from Fluorescent Microarrays White Paper Using TaqMan Endogenous Control Assays to Select an Endogenous Control for Experimental Studies (Aus) / (NZ)

84 Applied Biosystems,ABI PRISM and its design, and Primer Express are registered trademarks and Assays By Design and Assays on Demand, AB (Design), Applera and ROX are trademarks of Applera Corporation or its subsidiaries in the US and/or certain other countries AmpliTaq Gold and TaqMan are registered trademarks of Roche Molecular Systems, Inc. Applied Biosystems, All rights reserved The PCR Process and 5 nuclease process are covered by patents owned by Roche Molecular Systems, Inc. and F. Hoffmann-La Roche Ltd. For Research Use Only. Not for use in diagnostic procedures.

85 Thank You and Any Questions?

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