Significant difference or artefact of the method?
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1 Significant difference or artefact of the method? The impact of temperature performance of real-time thermocyclers on generated qpcr results Mary Span CYCLERtest B.V. Landgraaf The Netherlands
2 qpcr is a dynamic process qpcr is a dynamic reaction that works over a range of temperatures and concentrations highest conc. min. temp reaction optimum max. temp lowest conc. 11/03/09 2
3 Effect denaturation temperature and time 1s 5s 15 s 30 s 60 s The longer the denaturation time, the more inactivation of the Taq polymerase. Too low temperature, no denaturation. Too high temperature, lower yield. 11/03/09 3
4 Examples from the field High overshoot in fast mode: - Minimum overshoot 96,07 C - Maximum overshoot 101,32 C Difference between wells up to 5,25 C Non-uniformity up to 1,83 C sensor position (grey line) 11/03/09 4
5 Examples from the field Block non-uniformity increasing from 1,22 C to 2,29 C from beginning till end of plateau phase Lower overshoot in non-fast mode sensor (grey line) 11/03/09 5
6 Examples from the field Same thermocycler with aluminum block and with silver block Aluminium block: 1,11 C uniformity 3,22 C/s heat rate no overshoot Silver block is more uniform, faster heating, but therefore also showing overshoots Silver block: 0,58 C uniformity 4,06 C/s heat rate Average overshoot 96,37 C Maximum overshoot 97,15 C 11/03/09 6
7 Effect annealing temperature and time 1 s 5 s 15 s Longer annealing does not lead to higher or more specific yields. Too high temperature, lower yield. Too low temperature, non specific binding. 11/03/09 7
8 Examples from the field More than 2 C non-uniformity at annealing phase (primer pair designed with same Tm?) 11/03/09 8
9 Impact temperature on yield Annealing temperature too low Lower yield specific product, more non specific products, more primer dimers Primer dimers visible in a SYBR green assay In a Taqman assay only expressed as a lower Rn as primer dimers can not be detected 11/03/09 9
10 Impact non uniformity on melt curves Same qpcr reaction, only amplified and melted in different wells of a thermocycler Due to non-uniformity software registers peaks at different points in time and interprets them as different Tms dt=1,3 C 11/03/09 10
11 Impact non-uniformity on melt curves Up to 3,72 C non-uniformity during heating 11/03/09 11
12 High Resolution Melting Claims: Resolution up to 0,02 C (0,1 C on block cyclers) Facts: Block uniformity seldomly better than 0,5 C (in most cases 1 C and higher) during plateau phase During heating or cooling phase non-uniformity is even higher (typically between 3 and 7 C in block cyclers) How can a resolution of 0,1 C be achieved? Source: Corbett Research 11/03/09 12
13 Thermal variation uniformity accuracy Dots of the same color represent different models of thermocyclers of the same brand Size of the dots represents spread within population Data based on more than individual thermocyclers 11/03/09 13
14 Thermal variation Accuracy Model A Model B Two models of the same brand show a different accuracy and uniformity PCR which works on model A might fail on model B Every thermocycler outside the triangle will give inconsistent results using a kit which was optimized for 95 C with 2 C spread Dynamic Range of Kit Uniformity 11/03/09 14
15 Thermal variation The thermal non-uniformity of different models thermocyclers 11/03/09 15
16 Optical variation Source: Herman et al. Clinical Chemistry 52: (2006) The non-uniformity displayed in this graph is a combination of thermal non-uniformity plus optical non-uniformity 11/03/09 16
17 Controls in qpcr Type of controls used Positive and negative High positive, medium positive, weak positive, negative Positive dilution series, negative Internal controls Where would you put your positive and negative control? 11/03/09 17
18 Expression analysis In most methods to compare the expression of a gene of interest versus a reference gene a number of conditions have to be fulfilled. One of the most important ones is equal efficiencies of PCR How can all wells amplify with equal efficiency when in certain wells more Taq is inactivated than in others more primer dimers are formed than in others more non specific primers are formed than in others 11/03/09 18
19 Regulations in relation to diagnostic and molecular markers Most ISO norms used in diagnostic laboratories like ISO 17025, ISO and ISO do already require validation of thermocyclers at least once per year to exclude thermocycler artefacts Other organisations and Quality Programms like CAP (Pathology USA), New York State (Clinical Diagnostics), NFSCT (Forensics), AAFS, EFI (Immunogenetics Europe),ASHI (Immunogenetics USA), FELASA (Animal Health), AAVLD (Veterinary), Food Diagnostics, AACC, CPA (Pathology UK), GMP and GLP do also require validation of thermocyclers periodically. 11/03/09 19
20 Conclusions To do a reliable and reproducible qpcr it is not only necessary to pipet accurately and work with well optimized reagents, well designed oligos, pure and non-degraded DNA, optically clear plastics but also to control the thermal performance of the thermocycler Seen the variations in thermocyclers which are met in the field it is recommendable to check real time thermocyclers on a regular basis and to put at least controls on the coldest and hottest spots of a block Normalization for thermal and optical performance of the thermocycler and not just for reference genes is most ideal solution to exclude artefacts caused by the thermocycler 11/03/09 20
21 More infos /03/09 21
Tom Hendrikx**, Marc Verblakt*, Roger Pierik** * GENO-tronics BV, Landgraaf, The Netherlands ** CYCLERtest BV, Landgraaf, The Netherlands
The Impact of the Temperature Performance of Thermal (PCR) Cyclers on the Generated Results, and the Obligation for Regular Validation of PCR Thermal Cyclers Abstract Tom Hendrikx**, Marc Verblakt*, Roger
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