Novel In-process Monitoring Tools for Improving Accuracy and Reproducibility of RT-qPCR Results. Dirk Schacht, Global Product Manager, June 2015

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1 Novel In-process Monitoring Tools for Improving Accuracy and Reproducibility of RT-qPCR Results Dirk Schacht, Global Product Manager, June 2015 In-process monitoring tools for RT-qPCR 1

2 4-Part webinar series on gene expression workflow Part 1: Practical Hints and New Solutions for Successful Real-time PCR Studies Part 2: Novel In-process Monitoring Tools for Improving Accuracy and Reproducibility of qpcr Results Part 3: Critical factors for successful real-time multiplex PCR Part 4: Streamlining the Gene Expression Workflow while Increasing Data Quality

3 Legal Disclaimer 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 or can be requested from QIAGEN Technical Services or your local distributor. In-process monitoring tools for RT-qPCR 3

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 In-process monitoring tools for RT-qPCR 4

5 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 In-process monitoring tools for RT-qPCR 5

6 Why RT-qPCR Controls? 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 In-process monitoring tools for RT-qPCR 6

7 Various controls required for different aspects 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 In-process monitoring tools for RT-qPCR 7

8 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 In-process monitoring tools for RT-qPCR 8

9 Internal Control 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 kits or components False combination of reagents Human errors Preparation of reagents, master mixes, etc Reaction set up, cycler programming, etc IC (MAX) RNA (FAM) Cq shifts create greater problems than complete experiment failure In-process monitoring tools for RT-qPCR 9

10 Internal Control IC supports precise quantification results Spiked into sample at defined and reproducible concentration Identical amount in each experiment unlike reference gene Undergoes identical treatment as target RNA Duplex capacity in probe based procedures even increase precision Included in both reverse transcription and qpcr reactions Monitors complete 1-step or 2-step RT-PCR process Results in reproducible Cq values when used under same experimental condition Confirms inter-assay reproducibility QuantiNova IC RNA: a Cq deviation > 2 cycles indicates inhibition IC will give comparable results confirming experiments are comparable In-process monitoring tools for RT-qPCR 10

11 Internal Control QuantiNova IC RNA IC must detect presence of inhibitors at concentrations affecting normal transcripts Duplex one-step RT-PCR of HeLa RNA containing increasing amount of inhibitor In-process monitoring tools for RT-qPCR 11

12 Internal Control QuantiNova IC RNA IC should behave like normal transcripts in presence of inhibitors Two-step RT-PCR of RNA from total blood containing 0.003% SDS as inhibitor In-process monitoring tools for RT-qPCR 12

13 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 In-process monitoring tools for RT-qPCR 13

14 Other In-Process measures Additional options to improve RT-qPCR procedures and accuracy Integrated reduction or removal of genomic DNA Removes source of un-controlled Cq shift and misquantification Important if exon-spanning primers can not be used Hot Start procedures Avoids premature enzyme activity leading to non-specific PCR signal increase Allows room temperature or automated reaction set up for higher reproducibility QuantiNova Kits introduce 2-phase hot-start for both RT and qpcr reactions Visual pipetting control Prevents human errors Improves handling and work flow efficiency Integrated process measures remove variables and handling steps, avoiding potential inconsistency In-process monitoring tools for RT-qPCR 14

15 Removal of genomic DNA gdna can add to the specific transcript expression level, but can efficiently be removed Two-step RT-PCR of ACTB with and without removal of spiked gdna In-process monitoring tools for RT-qPCR 15

16 Reduction of genomic DNA gdna can add to the specific transcript expression level, but can efficiently be removed One-step RT-PCR of the single exon gene MGAT1 with and without reduction of spiked gdna In-process monitoring tools for RT-qPCR 16

17 Hot Start procedure in One-Step RT-qPCR Novel 2-phase hot-start activation of both reverse transcriptase and Taq polymerase Improved specificity and room temperature set up enabling automated handling In-process monitoring tools for RT-qPCR 17

18 Hot Start procedure in One-Step RT-qPCR Identical results after up to 2 hours room temperature storage of reaction set up One-step RT-PCR of various transcripts and amounts after up to 2 hours at 22 C Improved handling or automated reaction set up for higher precision and efficiency In-process monitoring tools for RT-qPCR 18

19 Built-in visual pipetting control Eliminate errors during reaction setup QuantiNova Master Mix contains 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

20 In-process control measures provide various advantages Process-integrated features increasing 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 can not be used 2-phase hot-start in RT-qPCR enabling room temperature set up Avoid premature enzyme activity and non-specific results Improve work procedures by room temperature or automated reaction set up Visual pipetting control Eliminate human errors Remove stress factor from staff In-process monitoring tools for RT-qPCR

21 In-process control measures provide various advantages New QuantiNova Kits for 1-step and 2-step RT-PCR include: target IC RNA Duplex capacity gdna treatment Room temp. set up QuantiNova Reverse Transcription Kit RNA, 2-step x n.a. x - - Visual pipetting 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 Q Product range providing process standards for 1-step and 2-step RT-qPCR In-process monitoring tools for RT-qPCR 21

22 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 In-process monitoring tools for RT-qPCR 22

23 Performance Data QuantiNova Reverse Transcription Kit Precise quantification over wide range of input amounts Two-step RT-PCR of serial dilution from 5µg to 10pg Leuko RNA used in cdna synthesis In-process monitoring tools for RT-qPCR 23

24 Performance Data QuantiNova Probe RT-PCR Kit Excellent performance and sensitivity compared to existing one-step RT-PCR kits One-step RT-PCR of 100ng 10pg HeLa RNA using TaqMan assays for MYC on CFX Connect (left diagram) and for CDKNB1 on ViiA7 (right diagram) In-process monitoring tools for RT-qPCR 24

25 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 In-process monitoring tools for RT-qPCR 25

26 Summary In-process control measures ensure confidence in RT-qPCR results Controls are an indispensable requirement for reliable transcript quantification Internal Control RNA provides safety measure on inhibition or performance issues gdna removal increases precision of transcript quantification 2-phase hot start improves specificity and handling in one-step RT-qPCR Visual pipetting control ensures correct and relaxed working steps New QuantiNova kits provide these in-process control measures with superior performance Ensure your insight on gene expression relies on facts, not on artefacts In-process monitoring tools for RT-qPCR 26

27 Thank you for attending Questions? Contact QIAGEN Technical Service Call: for US Call: for EU Dirk Schacht, Ph.D.

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