Agilent s Mx3000P and Mx3005P Realtime PCR just got better Dr. Ivan Bendezu Genomics Agent Andalucia
Real-time PCR Chemistries SYBR Green SYBR Green: Dye attaches to the minor groove of double-stranded DNA Very low fluorescence when not bound 1000x increase in fluorescence when bound to DNA Sequence unspecific: All double-stranded molecules are detected (specific amplicon, primer dimers etc.) SYBR green is a good beginners chemistry and most valuable in assay validation and for assay troubleshooting Page 2
Real-time PCR Chemistries Taqman Reporterdye Acceptordye (Quencher) unbound probe free in solution Taqman Chemistry: Uses exonuclease activity of Taq in combination with a dual-labeled oligonucleotide the probe. Light Taq energy transfer Light emission Higher specificity: Detection only if primer and probe are bound on the same strand Light Light emission Allows multiplexing of multiple target sequences in the same reaction Taq Page 3
Real-time PCR Instrumentation Emission filter wheel Hot top Excitation filter wheel Thermal system Tungsten/Halogen Lamp Mx3000P/Mx3005P Fiber optic cable and scanning arm Page 4
Real-time PCR Instrumentation 5-plex data 50 copies of target Dyes: Atto425, FAM, HEX, TxRd, Cy5 Built for multiplexing: Superior performance in multiplexing QPCR applications Flexible measurement options: ROX channel available for target detection as no reference dye is needed to correct for positional effects. Mx3005P is able to mismatch excitation and emission filters. Protein melt curve using SYPRO Orange (ex: FAM em: ROX) Excellent block uniformity: Highly uniform results across the block allow robust and low variance measurements for reliable and significant results Page 5
Real-time PCR Instrumentation Over 10 years of experience in real-time QPCR instrumentation Halogen lamp based excitation Freedom of choice: Choose the filter configuration that suits your needs. Use all available QPCR chemistries. Scanning optical design: Avoids positional effects 4 colors (Mx3000P ) or 5 colors (Mx3005P ) Photomultiplier tube allows sensitive measurement Internal computer saves data: Recovery of data in case of external computer failure Page 6
Real-time PCR Instrumentation Thermal system Temperature uniformity: (+/- 0.25 C @ 72 C across the block). Solid state, Peltier-based thermal system Ramp rate: up to 2.5 C/s Hot Top (Heated lid). others Mx3005 Variation of amplicon T m Herrmann et al., Clin. Chem. 52 (2006) and 53 (2007) Page 7
Real-time PCR Instrumentation - Optics Halogen lamp Excitation range 350-750 nm Excitation filter wheel 4 or 5 filters with narrow bandpass (~10 nm) Emission filter wheel 4 or 5 filters with narrow bandpass (~10 nm) Photomultiplier Highly sensitive detection of fluorescence Fibre optics cable Page 8
Simultaneous vs scanning illumination and detection Asynchronous (scanning) Constant intensity (No positional effects) Simultaneous (CCD) Intensity depends on well position Lamp Higher intensity lower intensity 3.5 s scanning time per dye
Excitation range: 350 to 750 nm Emission range: 350 to 700 nm Real-time PCR Instrumentation - Detected Dyes TET HEX TAM TxRd Alx350 Atto425 FAM JOECy3 ROX Cy5 Standard Available filter filters set can on Mx3005P Mx3000P be used with alternative dyes Freedom of choice: Choose the filter combinations that match your needs! Page 10
Real-time PCR Instrumentation - Optics Designed for multiplexing - Discrimination capabilities 350 nm 750 nm Excitation Filter 10 nm Interference Em. Filter 3000 2500 2000 RFU 1500 1000 500 0-500 FAM HEX ROX Cy5 FAM HEX ROX Cy5 Optimized Filter Sets Page 11
Adaptive Baseline Algorithm Adaptive baseline correction
Adaptive Baseline Corrects for Drift dr R
Moving Average Algorithm Random noise in baseline is removed to make it easier for sw to find exponential amp smoothed by replacing each data point with the average of the points around it. (user selectable, 3 pt default) Minor impact on Ct X
Optional Data Smoothing 3 Pt Smoothing
MxPro Software: Flexible and easy to use
MxPro Experiment Types Experiment types can be converted after data collection Probe based Quantification Gene expression Melt curve analysis Real-time AD/SNP analysis Determine opt annealing temp RNA and DNA quantitation End-point Allele Discrimination Now featuring Multiple experiment Analysis: Up to 12 plates!
Software Organization Follows Experimental Workflow
Run Status Box shows run time parameters
QPCR Assay QC & RNA Quality to Expression Levels (Application Note RNA QC/QPCR (5989-7730EN) GAPDH 3 assay: Expected size 126 bp oligo-dt random RIN 8.9 RIN 6.5 A B 0 min 30 min C RIN 4.6 D RIN 2.3 45 min 75 min 121 bp G A P D H 0-2 -4-6 -8-10 RIN 8.9 RIN 6.5 RIN 4.6 RIN 2.3 GAPDH 5' assay GAPDH 3' assay -12-14 DNA Chip to check QPCR amplicon product size 20 RIN # corresponding expression level changes in QRT-PCR
Real-time PCR Applications Versatile technology: Sensitive sequence detection and quantification Gene expression analysis Detection of sequence variants Fluorescence detection Pathogen detection GMO analysis Quality control Forensics Validation of microarrays Comparative quantification ChIP Copy number analysis CGH mirna detection SNP detection/ allelic discrimination Methylation studies Immuno-PCR: Detect proteins with QPCR DNA/RNA quantification Protein stability testing Reporter gene activity and many more.. Page 21
QPCR related reagent product portfolio AffinityScript : Multi temperature Reverse transcriptase Brilliant II MM: For SYBR green and probe. High Specificity mirna Detection kit: mirna detection by QPCR RNA extraction Reverse Transcription Controls QPCR Absolutely RNA : RNA extraction from cells and tissue SideStep : Cells to PCR Alien Inhibitor Alert: Inhibition control and external spike-in for normalization QPCR reference RNA: External reference sample for human and mouse Page 22
Absolutely RNA SideStep Ways of Working: Combining the strengths of 3 platforms Sample Preparation Nucleic acid quantification and QC Microarrays (expression, CGH, copy number) QPCR assay validation + Brilliant reagents QPCR (microarray validation, downstream studies) Page 23
Thanks for your attention! Page 24