the importance of controls and novel solutions for successful real-time qpcr
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Sample to Insight
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Novel In-process Monitoring Tools for Improving Accuracy and Reproducibility of RT-qPCR ResultsDirk Schacht, Global Product Manager, October 2015
In-process monitoring tools for RT-qPCR
Sample to Insight
Legal Disclaimer
Critical factors for successful real-time multiplex PCR 2
QIAGEN products shown here are intended for molecular biology applications. These products are not intended for the diagnosis, prevention or treatment of a disease.
For up-to-date licensing information and product-specific disclaimers, see the respective QIAGEN kit handbook or user manual. QIAGEN kit handbooks and user manuals are available at www.QIAGEN.com or can be requested from QIAGEN Technical Services or your local distributor.
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In-process monitoring tools for RT-qPCR 3
Agenda
Controls for RT-qPCR in gene expression analysis
Internal Control RNA in gene expression analysis
Other useful in-process safety measures
Performance data of new QuantiNova Reverse Transcription and Probe RT-PCR Kits
Summary and Q&A
Sample to Insight
In-process monitoring tools for RT-qPCR 4
Agenda
Controls for RT-qPCR in gene expression analysis
Internal Control RNA in gene expression analysis
Other useful in-process safety measures
Performance data of new QuantiNova Reverse Transcription and Probe RT-PCR Kits
Summary and Q&A
Sample to Insight
In-process monitoring tools for RT-qPCR
Why RT-qPCR controls?
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Standardization ensures accuracy and reproducibility of experimental results
Confirm validity of experimental design
Reduce variables to avoid artifacts
Increase predictive value of research
Provide evidence concordant with GLP, MIQE* guidelines, etc.
Ensure your findings and insight are relevant * Bustin, A.S. et al. (2009) The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments. Clinical Chemistry, 55, 611.
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In-process monitoring tools for RT-qPCR
Various controls required for different aspects
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Commonly used controls in RT-qPCR
No-template control Detects reagent or equipment contamination => confirms positive results
No-RT or gDNA contamination control Detects signals generated from gDNA => confirms transcript specific signal
No-amplification control Detects background fluorescence generated by degraded dual-labelled probes
Positive control or internal control Detects inhibitors or malfunction => confirms reagents and equipment are working
Reference genes or quantification calibrators Not “controls” Used in relative or absolute quantification
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In-process monitoring tools for RT-qPCR 7
Agenda
Controls for RT-qPCR in gene expression analysis
Internal Control RNA in gene expression analysis
Other useful in-process safety measures
Performance data of new QuantiNova Reverse Transcription and Probe RT-PCR Kits
Summary and Q&A
Sample to Insight
In-process monitoring tools for RT-qPCR
Internal Control
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Internal Control (IC) ensures precise quantification and inter-sample consistency
Internal Control detects Inhibitor contamination
From sample (heme, urea, etc.) From extraction method (alcohol, salt, phenol, etc.)
Instrument malfunction Temperature calibration Program errors
Chemistry failures Expired or degraded kit or components False combination of reagents
Human error Preparation of reagents, master mixes, etc. Reaction setup, cycler programming, etc.
Cq shifts create more problems than complete experiment failure
RNA (FAM)
IC (MAX)
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In-process monitoring tools for RT-qPCR
Internal Control
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IC supports precise quantification results
Spiked into sample at a defined and reproducible concentration Identical amount in each experiment – unlike reference gene
Undergoes identical treatment as target RNA Duplex capacity in probe-based procedures – increased precision
Included in both reverse-transcription and qPCR reactions Monitors complete 1-step or 2-step RT-PCR processes
Results in reproducible Cq values when used under the same experimental condition Confirms inter-assay reproducibility QuantiNova IC RNA: a Cq deviation >2 cycles indicates inhibition
IC will give a similar result confirming that experiments are comparable
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In-process monitoring tools for RT-qPCR
Internal Control – QuantiNova IC RNA
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IC must detect presence of inhibitors at concentrations affecting normal transcripts
Duplex 1-step RT-PCR of HeLa RNA containing increasing amount of inhibitor
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In-process monitoring tools for RT-qPCR
Internal Control – QuantiNova IC RNA
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IC should behave like normal transcripts in the presence of inhibitors
2-step RT-PCR of RNA from total blood containing 0.003% SDS as an inhibitor
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In-process monitoring tools for RT-qPCR 12
Agenda
Controls for RT-qPCR in gene expression analysis
Internal Control RNA in gene expression analysis
Other useful in-process safety measures
Performance data of new QuantiNova Reverse Transcription and Probe RT-PCR Kits
Summary and Q&A
Sample to Insight
In-process monitoring tools for RT-qPCR
Other in-process safety measures
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Additional options to improve RT-qPCR procedures and accuracy
Integrated reduction or removal of genomic DNA Removes source of uncontrolled Cq shift and misquantification Important if exon-spanning primers cannot be used
Hot-start procedures Avoids premature enzyme activity leading to nonspecific PCR signal increase Allows room-temperature or automated reaction setup for higher reproducibility QuantiNova Kits introduce 2-phase hot-start for both RT and qPCR reactions
Visual pipetting control Minimizes human error Improves handling and workflow efficiency
Integrated in-process safety measures remove variables and handling steps, avoiding potential inconsistency
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In-process monitoring tools for RT-qPCR
Removal of genomic DNA
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gDNA can contribute to the specific transcript expression level, but can efficiently be removed
2-step RT-PCR using ACTB primers, with and without removal of spiked gDNA
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In-process monitoring tools for RT-qPCR
Reduction of genomic DNA
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gDNA can contribute to the specific transcript expression level, but can efficiently be removed
1-step RT-PCR of the single exon gene MGAT1, with and without reduction of spiked gDNA
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In-process monitoring tools for RT-qPCR
Hot-start procedure in 1-step RT-qPCR
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Novel 2-phase hot-start activation of both reverse transcriptase and Taq DNA Polymerase
Improved specificity and room-temperature setup enabling automated handling
:
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In-process monitoring tools for RT-qPCR
Hot-start procedure in 1-step RT-qPCR
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Identical results after up to 2 hours of room-temperature storage
1-step RT-PCR of various transcripts and amounts after up to 2 hours at 22°C
Improved handling or automated reaction setup for higher precision and efficiency
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Built-in visual pipetting control
Minimize errors during reaction setup
QuantiNova Master Mix contains an inert blue dye
Yellow Template Dilution Buffer, used to dilute the template nucleic acid, contains an inert yellow dye
These dyes do not interfere with real-time PCR
Color changes from blue to green when adding template
Particularly useful for 96- or 384-well format
Ensures accurate reaction setup without impacting performance
In-process monitoring tools for RT-qPCR
Sample to Insight
In-process safety measures provide many advantages
Integrated controls increase accuracy and reproducibility in gene expression profiling
Internal Control RNA Monitor and confirm performance of RT and qPCR reactions Rule out potential sources of artifacts
Reduction or removal of genomic DNA Improve precision and reproducibility in transcript quantification Reliable results if exon-spanning primers cannot be used
2-phase hot-start in RT-qPCR enables room-temperature setup Avoid premature enzyme activity and nonspecific results Improve work procedures by room-temperature or automated reaction setup
Visual pipetting control Minimize human error Remove stress factor
In-process monitoring tools for RT-qPCR
Sample to Insight
In-process monitoring tools for RT-qPCR
In-process control measures provide various advantages
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New QuantiNova Kits for 1-step and 2-step RT-qPCR include:
Product range provides process standards for 1-step and 2-step RT-qPCR
Target IC RNA Duplex capacit
y
gDNA treatment
Room-temp. setup
Visual pipetting
QuantiNova Reverse Transcription Kit RNA, 2-step x n.a. x – –
QuantiNova Probe PCR Kit cDNA, 2-step – x n.a. x x
QuantiNova SYBR Green PCR Kit cDNA, 2-step – n.a. n.a. x x
QuantiNova Probe RT-PCR Kit RNA, 1-step x x x x x
QuantiNova SYBR Green RT-PCR Kit * RNA, 1-step x n.a. – x x
* Coming soon!
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In-process monitoring tools for RT-qPCR 21
Agenda
Controls for RT-qPCR in gene expression analysis
Internal Control RNA in gene expression analysis
Other useful in-process safety measures
Performance data of new QuantiNova Reverse Transcription and Probe RT-PCR Kits
Summary and Q&A
Sample to Insight
In-process monitoring tools for RT-qPCR
Performance data – QuantiNova Reverse Transcription Kit
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Precise quantification over a wide range of input amounts
2-step RT-PCR of a serial dilution of Leukocyte RNA (5 µg – 10 pg) used in cDNA synthesis
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In-process monitoring tools for RT-qPCR
Performance data – QuantiNova Probe RT-PCR Kit
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Excellent performance and sensitivity compared to existing 1-step RT-PCR kits
1-step RT-PCR using HeLa RNA (100 ng – 10 pg) and TaqMan assays for MYC on a CFX Connect (left) and for CDKNB1 on ViiA7 (right)
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Agenda
Controls for RT-qPCR in gene expression analysis
Internal Control RNA in gene expression analysis
Other useful in-process safety measures
Performance data of new QuantiNova Reverse Transcription and Probe RT-PCR Kits
Summary and Q&A
Sample to Insight
In-process monitoring tools for RT-qPCR
Summary
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In-process control measures ensure confidence in RT-qPCR results
Controls are an indispensable requirement for reliable transcript quantification
Internal Control RNA indicates inhibition or performance issues
gDNA removal increases precision of transcript quantification
2-phase hot-start improves specificity and handling in 1-step RT-qPCR
Visual pipetting control ensures correct and relaxed working steps
New QuantiNova Kits provide these in-process control measures for superior performance
Ensure your gene expression insights are based on facts, not artifacts
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Questions?
Thank you very much for your attention!
Dirk [email protected]
For up-to-date licensing information and product-specific disclaimers, see the respective QIAGEN kit handbook or user manual. QIAGEN kit handbooks and user manuals are available at www.QIAGEN.com or can be requested from QIAGEN Technical Services or your local distributor.
In-process monitoring tools for RT-qPCR