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

Sample to Insight

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

Sample to Insight

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!

Sample to Insight

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)

Sample to Insight

In-process monitoring tools for RT-qPCR 24

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 Schachtdirk.schacht@qiagen.com

qiawebinars@qiagen.com

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