Scott Reierstad. Field Applications Scientist

Transcription

1 Real-time PCR Training Scott Reierstad Field Applications Scientist

2 Topics we ll cover Quick introduction to real-time PCR ABI-supported real-time chemistries Instrumentation Quantitation via Standard Curves Relative Gene Expression workflow Protein Thermal Shift Live Software Demo 2

3 3 ViiA 7 Real-Time PCR System

4 96, 96 FAST, 384, TLDA thermal blocks Block change in less than 1 min Heated Cover (96 vs 384) Plate Adaptor (96 vs 384 vs TAC) 384-well and TAC Blocks at launch Validated Programmable uL 1-30uL TAC 1uL 1uL 96-well and Fast 96-well followed Validated Programmable uL 1-200uL Fast uL 1-100uL 4 Life Technologies Proprietary & Confidential 4/10/2013

5 Touchscreen: Main Screen Run experiments directly from touchscreen Collect ran experiments Set shortcuts from the home screen In summary: you do not need to have a PC* connected to the instrument to run an experiment *provided 5 Life Technologies Proprietary & Confidential 4/10/2013

6 User Experience 100+ plate studies without a database Streamlined Importing sample info and exporting run results Intuitive software (similar to StepOnePlus and 7500 v2.0 Upload 7900 Protocols / Export 7900 formats Monitor run remotely 6

7 ViiA 7-Twister II Flexible: multiple ViiA 7s to 1 robot All Plates Able to use FAST plates 2 input, 1 output 80 plate input 7 Life Technologies Proprietary & Confidential 4/10/2013

8 The Optics: OptiFlex System 6 Excitation and 6 Emission filters (455nm to 722nm) Excitation and Emission Filters can be decoupled for optimal flexibility FAM, SYBR, SYTO9 (MeltDoctor), Fluorescein, Sypro Orange, LC Green, Eva Green, Pulsar 650 VIC, JOE, TET, HEX TAMRA, NED, CY3, Bodipy TMR-X ROX, Texas Red Liz, CY5 Cy5.5, Alexa, Joda-4 8 Life Technologies Proprietary & Confidential 4/10/2013

9 Expanded Multiplex Capabilities 9 Multiplex on cdna with 5 probes

10 Dynamic Range, Fast Run Amplification of plasmid DNA (7-7E10 total copies/rxn) using the 384-well block. 9 logs of linear dynamic range in 35 minutes. 10 Life Technologies Proprietary & Confidential

11 ViiA 7: 1.5 Fold Sensitivity RNaseP copies: - 10,000-6,667-4,500-3,000-1,500-1,000 11

12 12 Calibration

13 Calibration Hardware: Requires Calibration plates ROI, Background, Uniformity, Pure Dyes, Normalization, HRM (optional), Verify instrument performance using RNaseP Calibrations: Takes about 10 min/ per plate Performed by install User recommended to re-calibrate every 6 months. How to calibrate: Access via instrument console, select instrument from My Instruments Click on instrument to access Instrument Manager 13

14 Custom Dye Calibration Dye MUST be added to dye library before performing a custom calibration Default temperature = 60 C 14

15 Setting Calibration Reminders Select the instrument from the Instrument Console Go to Calibration Reminders Address Settings 15

16 Maintenance Schedule Frequency Weekly Action 1. Power cycle instrument controlling computer 2. Perform instrument self test Monthly 1. Check lamp status with software 2. Perform background calibration Every 6 mos 1. Run instrument calibration kit (ROI, Background, Dye, Normalization, RNaseP 16

17 System normalization Following each real-time run, we will carry out normalization steps to compensate for two different variables: Non-PCR fluctuations in fluorescence. Background signal. 17

18 Common sources of fluorescent variation Light source optics cover condensation 18

19 ROX Passive Reference Dye Greatly improves precision of replicates. (Reporter) (Rox) 10 ng Sample A Well 1 Well 2 Normalized reporter = Reporter / Rox Rn 19

20 ROX dye better precision 36 replicates analyzed with ROX passive reference dye. 36 replicates analyzed without ROX. 20

21 Two points about ROX All ABI real-time PCR master mixes contain ROX. ABI software automatically performs normalization to ROX, unless you specifically tell it not to. If you do not use ROX, you must disable the passive reference in software for accurate analysis. 21

22 Amplification plot graphs cycle vs. fluorescence? Fluorescent units 22

23 After we account for non-pcr variables properly, we obtain Ct values BUT, we still need some way to convert Cts into numbers we can make sense of. Two ways... 46

24 Can use a standard / dilution curve Do serial dilutions

25 At the end of your real-time run 10, Qty = 250 Standard / dilution amt Unknown sample Ct Cycle (Ct) 48

26 Two curve types Absolute standard curves Actual copy number of standard is known. Ex., microbial quantification. 49

27 Absolute curve absolute copies Qty = 25,054 Standard / dilution amt Unknown sample Ct Cycle (Ct) 50

28 Two curve types Absolute standard curves Actual copy number of standard is known. Ex., microbial quantification. Dilution (a.k.a., relative standard) curve Only dilution factor is known. Ex., Gene expression, copy number. 51

29 52 What if you hate standard / dilution curves?

30 Reasons some don t like standards Take up too much room on reaction plate. Extra reagent cost. Time-consuming to prepare. Difficult to pipet accurately. 53

31 Example experiment I have 3 servings of bean sprouts of equal mass. My concern: sprouts contaminated with E. coli. I isolate DNA from each. Next, I design an E. coli-specific TaqMan Assay. Now, I amplify DNA from all three in real-time. Note: There are no curves on this reaction plate. 54

32 Real-time results Sample 1 Sample 2 Sample 3 Cycle number 55

33 Q: Can I say anything about starting amounts? Sample 1 Sample 2 Sample 3 Relatively speaking... Yes! Cycle number 56

34 PCR should double product after each cycle in geometric phase Sample 1 Sample 2 Sample 3 Cycle number Ct=1 Two-fold difference 57

35 Handy relationship for calculating relative starting amounts Ct of 1 = 2-fold difference Ct of 2 = 4-fold difference Ct of 3 = Ct of = fold difference 10-fold difference However, math makes an assumption... 58

36 Math makes an assumption Namely, that we really are doubling the amount of product with each cycle. -Doubling = 100% amplification efficiency* *Efficiency: the percentage of target molecules used as template for each round of geometricphase amplification. 59

37 When we have 100% efficiency 100% 10-fold = 3.3 cycles

38 However, if we reduce efficiency % 10-fold = 3.3 cycles % 10-fold = 3.9 cycles

39 Relative Quantification (gene expression): Relative quantification is used to determine fold differences of a target nucleic acid (usually RNA) between two samples. Ex: Gene expression 109

40 Most common method for Relative Quantification is the Ct method (does not require standard cuves): At least two samples: Test sample and a calibrator/reference sample. This is a sample to which unknown samples are compared (example -untreated sample, control, timepoint zero ). At least two genes: Target gene and Endogenous Control. This is a transcript that is present at a constant amount in total RNA (housekeeping gene). Levels are used to normalize differences in the amount of total RNA loaded in each reaction. 110

41 Sample experiment: IL-4 expression change following drug treatment Calibrator/Ref sample t=0 t=12 t=24 t=48 time total RNA total RNA total RNA total RNA cdna cdna cdna cdna 111 Life Technologies Proprietary & Confidential 4/10/2013

42 Comparison of Target Gene and Endogenous Control Rn Ct =14 Ct = 24 Cycles 112 Life Technologies Proprietary & Confidential 4/10/2013 Endogenous control (18S) Target gene (IL-4)

43 Comparative Ct Method R n t=0 R n t=12 h Ct=23 Ct=30 Cycle s Ct=22 Ct=27 Cycle s R n t=24 h R n t=48 h Ct=24 Ct=26 Cycle s Endogenous control Target gene Ct=23 Ct=33 Cycle s 113 Life Technologies Proprietary & Confidential 4/10/2013

44 Fold change calculation-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 - Fold Change = Ct 114 Life Technologies Proprietary & Confidential 4/10/2013 The software does this automatically

45 Best part about ViiA 7 software? It does all the math for you! 115

46 116

47 ddct method (fold changes) is fast and easy but works only if: 2 - Ct Efficiencies (E) are equal! Target gene Endogenous Efficiencies (E) of genes are 100% Qf = Qs (1+Eff) C 117

48 In Ct method efficiencies MUST be equal (+/- 10%)!!! Ct Efficiency test Ct Ct Ct value IL-4 Log of Input 18S Log of Input slope Ct allowed 118

49 What if efficiencies are not equal? Relative standard curve method (long, tedious) -Must use a standard curve on each plate for all genes -Fold changes are calculated using values calculated on a standard curve Efficiency correction* (ViiA 7 can do this) -Must use a standard curve one time only for all genes -Fold changes are calculated using a mathematical formula factoring different efficiencies *Nucleic Acids Research, 2001, Vol. 29, N.9 119

50 Efficiency correction: alternative to relative standard curve method (Pfaffl efficiency correction method*) 120 Life Technologies Proprietary & Confidential 4/10/2013 *A new mathematical model for relative quantification in real-time RT PCR Nucleic Acids Research, 2001, Vol. 29, N.9

51 Multiple endogenous control normalization (gnorm algorithm*) Vandesompele J, et. al: Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biology, 2002, June Life Technologies Proprietary & Confidential 4/10/2013

52 Expression Suite Software Free data analysis software from AB for ddct data analysis. Accepts run files (one or multiple runs), then calculates RQ data. Numerous plots and features: heat maps, volcano plots, endogenous control selection, etc. To locate and download, go to AB s website and search for Expression Suite. 122

53 Protein Thermal Shift Solution Using Applied Biosystems Real- Time PCR Systems

54 Training Outline Application Overview Product Offering Overview Protein Thermal Shift workflow: Set up a run Assay setup and optimization Data Examples Live Software Workflow Demo: Setup, analysis, export 124

55 Protein Thermal Shift Software and Reagents Application Overview 125 4/10/2013 Life Technologies Proprietary and confidential

56 Protein Thermal Shift Is a technique used to study the thermal stability of a protein using melt curves in Real Time PCR. Many applications Protein Stability Screen: improving protein preps (ph, salt, excipients) profiling crystallization conditions protein formulation and storage buffers effect of mutations or modifications Protein prep QC High-Throughput Ligand Screening: Small-molecule and fragment screens Antibody-target specificity Protein-protein interaction Inhibitor binding 126

57 How does Protein Thermal Shift work? The protein unfolds as it is heated. An environmentallysensitive dye binds exposed hydrophobic regions and fluoresces. The Tm (melting temperature) is calculated from the melt curve. Changes in Tm are correlated to changes in protein stability. Fluorescence Temperature ( o C) Calculate the inflection point of the curve 127

58 Complete Protein Thermal Shift Solution 1 µg Protein/Well Applied Biosystems Applied Biosystems PTS + PTS Dye Real-Time Instrument Analysis Software PTS Advantages : High Throughput: 384 assays in < 30 minutes, robotics available <1 ug protein / assay No protein or ligand structure information necessary Minimal upfront optimization 128

59 Protein Thermal Shift Software and Reagents Product Offering Overview 129 4/10/2013 Life Technologies Proprietary and confidential

60 Applied Biosystems Total Protein Thermal Shift Solution Optimized Protein Thermal Shift reagents: Starter Kit: Control Protein + Ligand (100 rxns), Protein Thermal Shift Dye and Buffer (2000 rxns) Dye Kit: Protein Thermal Shift Dye and Buffer (2000 rxns) Streamlined software workflow Accepts *.eds files from StepOne, StepOnePlus, 7500 Fast, ViiA 7 and QuantStudio 12K Flex Systems. Multi-plate study-based software (>100 x 384-well plates/study) No special calibration required, uses ROX dye calibration Calculate Tm with respect to a Reference Sample across plates 130

61 Protein Thermal Shift Software v1.1 Life Technologies owns the exclusive rights to the Protein Thermal Shift application. Stand-alone, Protein Thermal Shift analysis software Compatible with *.eds Melt run files from: StepOne Software v2.2 (or later) 7500 Software v2.0.4 (or later) ViiA 7 Software v1.0 (or later) QuantStudio 12K Flex Software v1.0 (or later) Brand new analysis application Multi-plate study-based analysis for High Throughput experiments Define assay conditions and setup within the software Single/Multiple peak and Auto/Manual analysis options Boltzmann fit and Derivative Analysis results Calculate Tm and Delta Tm ( Tm) 131

62 Protein Thermal Shift Software and Reagents Workflow 132 4/10/2013 Life Technologies Proprietary and confidential

63 Protein Thermal Shift basic workflow Run melt experiment on AB qpcr Instrument Analyze the experiment on the qpcr instrument sw Open or Start Study in Protein Thermal Shift software Use ROX Detector No Passive Reference Protein Thermal Shift software will only accept analyzed *.eds files A Study is a collection of runs from a single instrument platform Import.eds file(s) into Study Studies can contain >100 *.eds run files Analyze melt curves in Protein Thermal Shift software Each Analysis Group contains a single Reference sample group 133

64 Protein Thermal Shift Software Basic Workflow Run Setup The ramp rate can be made faster for shorter runs, but some detail for the melt will be lost. Researchers need to optimize their melt conditions for individual proteins and screens. 134 Life Technologies 4/10/2013

65 Assay Setup and Optimization Assay Optimization can be performed using the PTS Dye (1X-20X) and various amounts of protein Use ul total volume per reaction Use ~0.05-5µg (on average 1ug) of protein per reaction Setup reaction on ice: protein, Protein Thermal Shift Dye, buffer and compound. Start run as soon as convenient. Dye stability for the assay at 4 C as been tested to 24 hrs, at Room Temperature in the dark also 24 hrs. Perform continuous dissociation with 1-6% ramp rate (obtain results in minutes) or C/second 135

66 Assay Optimization Workflow Conditions to vary: Protein: ug per well Dye: 1X-20X ph: ph 2.0 ph 9.0 Salt: protein dependent Buffers: We have successfully tried HEPES, Tris, KPO4, NaCitrate, Glycine HCl Ligands: small molecules and fragment libraries. 136

67 Guidelines to consider Use a protein that has Tm < 98 C. In the native state, ensure that the protein has no external hydrophobic sites and that it has sufficient internal hydrophobic residues. Multi-domain proteins, or proteins that form oligomers may undergo a multi-state unfolding and multiple melt phases in a melt curve. All of these conditions may be tweaked by performing a buffer/additive screening study so that the protein unfolds in a 2-state model. Titration study of PTS dye and Protein may be necessary to assess optimal protein:dye ratio Ramp speed and ramp rate can be optimized to achieve optimal data resolution Recommended that each plate has a reference group Recommended that each protein melt reaction is repeated 4 times on a plate to ensure statistical significance. 137

68 Other guidelines to consider The PTS software is used to simply get Tm of a reverse melt curve data, and have ability to create melt curve studies. Keep things simple; 95% of the software is simply assigning wells (colorcoding/sorting/grouping melt curve data). The final summary of results is captured in the Replicate Results table. The Tm s that are provided are the Tm B (Boltzmann; based on a sigmoidal curve of raw data) and Tm D (derivative). 138

69 Other guidelines to consider 2 Tm s are provided Tm B (Boltzmann; based on a sigmoidal fit of raw data) Tm D (derivative) X1 M3 filter (Viia7 only) 139

70 Guidelines and Recommendations cont. NPC: Contains only buffer, water, and dye LOC: Contains only ligand, buffer, water, and dye. Ligands are capable of skewing data if ligand interacts with dye. Prepare a fresh dilution of Protein Thermal Shift Dye (1000 ) to 8. Keep reaction cold at all times prior to putting it on the machine. PTS software runs an algorithm that reduces the noise in the primary raw melt curve data file. So curves may look a little different. 140

71 Protein Thermal Shift Software and Reagents Example Data 141 4/10/2013 Life Technologies Proprietary and confidential

72 Protein-Ligand Binding: Increase in Protein Thermal Stability with Bound Ligand Melt Curve Protein Protein + Ligand 41 o C No Protein Control 48 o C Derivative Curve ~ 7 o C delta Tm ViiA TM 7 Real Time PCR System 142

73 Effect of Buffer Conditions on Protein Thermal Stability Higher Tm Protein Stability -Na citrate ph mm NaCl -KPO 4 ph mM NaCl -KPO 4 ph mM NaCl -Hepes. ph mM NaCl Better Buffer! 143

74 Examine effects of point mutations & ligand binding on protein stability WT, Mut1, Mut2 WT, Mut1, Mut2 + Ligand 43.8 o C 49.2 o C 55.5 o C 46.3 o C 51.1 o C 57.0 o C PTS data from StepOnePlus TM instrument showing the Normalized Reporter and Derivative Melt profiles 144

75 Conclusions The Protein Thermal Shift Assay is a rapid, inexpensive, and straightforward high-throughput tool for screening conditions that maximize protein stability or libraries of ligands. PTS has been performed on many Applied Biosystems Real-Time PCR Instruments, expanding the flexibility of these systems to protein research. Applied Biosystems complete Protein Thermal Shift solution: dye reagent instrumentation analysis software 145

76 162 Where can I find technical help?

77 163 Getting Started Guides

78 164 ABI online support

79 165 ABI online support

80 Technical Support Hotline

81 Scott Reierstad, Field Applications Scientist 167

82 Trademarks For Research Use Only. Not for diagnostic procedures. The PCR process and 5' nuclease process are covered by patents owned by Roche Molecular Systems, Inc. and F. Hoffmann-La Roche Ltd. Applied Biosystems, ABI (Design), and VIC are registered trademarks, and Applera and FAM are registered trademarks of Applera Corporation or its subsidiaries in the US and/or certain other countries. TaqMan and AmpliTaq Gold are registered trademarks of Roche Molecular Systems, Inc. SYBR Green is a registered trademark of Molecular Probes, Inc. All other trademarks are the sole property or their respective owners Applied Biosystems. All rights reserved. 168