One Step Ahead for Your RT-PCR

One Step Ahead for Your RT-PCR
Sample to Insight One Step Ahead for Your RT-PCR Dr. Bernd Willems, Global Product Manager 1
Sample to Insight Legal disclaimer One Step Ahead for Your RT-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.
Sample to Insight Agenda 3 Introduction OneStep Ahead RT-PCR: Eight pitfalls and one solution How to use the kit Recommendations Summary One Step Ahead for Your RT-PCR 1 2 3 4 5
Sample to Insight Introduction 5 What is a one-step RT-PCR? A shortcut in your workflow One Step Ahead for Your RT-PCR
Sample to Insight Introduction 6 The advantages of one-step RT-PCR • Streamlined workflow • Save time / effort / money • One-tube format reduces risk of contamination • More robust and reliable results One Step Ahead for Your RT-PCR
Sample to Insight Eight pitfalls and one solution 8 1. Influence of annealing temperature on specificity 2. Nonspecific amplification during reaction setup 3. Risk of RNase contamination 4. PCR-borne mutations 5. RNA secondary structure and high GC-content 6. Pipetting errors 7. False negative results 8. Target RNAconcentration threshold Eight pitfalls for all who are performing one-stepRT-PCR One Step Ahead for Your RT-PCR
Sample to Insight Eight pitfalls and one solution 9 Pitfall one:Influence of annealing temperature on specificity The QIAGEN OneStep Ahead RT-PCRkit’s solution: K+ and NH4+ -unique dual cation formulation One Step Ahead for Your RT-PCR
Sample to Insight Eight pitfalls and one solution 10 Key technologies to improve annealing specificity: The buffer K+ and NH4+ -unique dual cation formulation: Stabilization by potassium cations interacting with the phosphate backbone Destabilization by ammonium cations interacting with the hydrogen bonds of nucleotide mismatches Pitfall one:Influence of annealing temperature on specificity (cont’d) One Step Ahead for Your RT-PCR
Sample to Insight Eight pitfalls and one solution 11 Key technologies to improve annealing specificity: The buffer K+ and NH4 + -unique dual cation formulation: Thus, only specific binding remains stable… …which guarantees the highest degree of specificity Pitfall one:Influence of annealing temperature on specificity (cont’d) One Step Ahead for Your RT-PCR
Sample to Insight Eight pitfalls and one solution 12 Indicated amounts of HeLa total RNA (in ng) w ere used as template for amplification of EIF2B4 (107 bp) in duplicate, according to the suppliers’ instructions. Green arrow s indicate specific product, red arrow s indicate primer‐dimers, purple arrow s indicate nonspecific amplification products. Analysis w as performed using the QIAxcel. Pitfall one:Influence of annealing temperature on specificity (cont’d) Comparing the QIAGEN Kit performance with competitors One Step Ahead for Your RT-PCR
Sample to Insight Eight pitfalls and one solution 13 One annealing temperature for all assays. 45°C 60°C Many assays work over an annealing temperature range from 45-60°C. -55°C proofed to be a universally recommendable annealing temperature. Pitfall one:Influence of annealing temperature on specificity (cont’d) One Step Ahead for Your RT-PCR
Sample to Insight Eight pitfalls and one solution 14 Pitfall two:Nonspecific amplification during reaction setup • Residual activity of RT enzyme at room temperature leads to unspecific amplification at primer dimers • These are competing for resources with the target of interest The QIAGEN OneStep Ahead RT-PCRkit’s solution: • The unique RT-Blocker keeps the RT-enzymes completely inactive at ambient temperatures • It only dissociates to activate the RT-enzyme when temperature reaches the catalytic optimum for reverse transcription • This enables convenient room temperature setup and even allows keeping the reactions at room temperature for up to 2h before cycling • This enables use of the kit in automated robotic workflows One Step Ahead for Your RT-PCR
Sample to Insight Eight pitfalls and one solution 15 Pitfall two:Nonspecific amplification during reaction setup (cont’d) One Step Ahead for Your RT-PCR
Sample to Insight Eight pitfalls and one solution 16 Pitfall three:Risk of RNAse contamination • RNAs are highly fragile molecules; RNA cleaving RNases are widely dispersed in our environment • Rnases can easily contaminate your sample material and destroy precious RNA The QIAGEN OneStep Ahead RT-PCRkit’s solution: • Included RNase inhibitor prevents RNA decay caused by accidental RNase contamination • One-step RT-PCR format ensures minimal contamination due with a one-tube approach One Step Ahead for Your RT-PCR
Sample to Insight Eight pitfalls and one solution 17 Pitfall four:PCR-borne mutations • PCR amplification using normal Taq DNA polymerase is prone to error in the DNA replication process • Typical rate of point mutations caused by Taq is 1 in 9,000 The QIAGEN OneStep Ahead RT-PCR kit’s solution: • An additional high-fidelity enzyme with a 3' to 5' exonuclease proofreading activity elevates the overall fidelity of the PCR step • Furthermore it improves the processivity to allow for amplification of longer targets up to 4kb HeLa total RNA (100 and 10 ng) w as used as a template for amplification of RANBP2 (3 kbp). One Step Ahead for Your RT-PCR
Sample to Insight Eight pitfalls and one solution 18 Pitfall five:RNAsecondarystructure and high GC-content QIAGEN Supplier T • RNA with complex secondary structure can cause the reverse transcriptase to stop or dissociate from the RNA template • Furthermore, the reverse transcriptasecan skip over looped-out regions of RNA • GC-rich sequences cause tight association of RNA:DNA hybrids which interfere with primer binding and thus prevent amplification The QIAGEN OneStep Ahead RT-PCRkit’s solution: • The combination of Omniscript and Sensiscript ensure highest affinity to any RNA template • Q-Solution facilitates reverse transcription and amplification of templates with a high GC-content or a high degree of secondary structure • Often enables or improves a suboptimal PCR caused by templates that have a high degree of secondary structure or that have a GC-rich sequence • Used at just one working concentration,which has been optimized for the requirements of multiplex PCR Figure 3: HeLa total RNA (100 ng) was used as template for amplification of TNFRI (581 bp) with a GC-ratio of 67.1%. Reactions were performed in triplicate, according to the suppliers’ instructions. Green arrows indicate specific product. Q-Solution was used for the QIAGEN reactions. One Step Ahead for Your RT-PCR
Sample to Insight Eight pitfalls and one solution 19 Pitfall six: Pipetting errors • A simple yet very effective solution: Optional tracking system for visual indication of correct pipetting • Consists of two inert dyes, a yellow Master Mix Tracer and a blue Template Tracer which can be added to the master mix and the template, respectively • When the template is pipetted into the master mix, the solution turns from yellow to green • Both dyes also serve as gel tracking dyes during electrophoresis. • Pipetting errors are a common problem in daily lab routine • When pipetting colorless solutions on a large scale (e.g. 96 well plates), keeping track is difficult The QIAGEN OneStep Ahead RT-PCR kit’s solution: One Step Ahead for Your RT-PCR
Sample to Insight 20 Color switch after template addition 1 1 2 3 4 1: no template added 2: 14 µl template added 3: 5 µl template added 4: 2 µl template added 1 1 2 3 4 10 µl of 25 µl reaction loaded 1,5% agarose gel ~ 50 bp ~ 4 kbp 1 2 3 4 Eight pitfalls and one solution Template Master Mix One Step Ahead for Your RT-PCR
Sample to Insight Eight pitfalls and one solution 21 Pitfall seven: False negative results • Control experiments are necessary to tell whether the absence of a PCR product can be interpreted as a negative result or an unsuccessful PCR • Full proof of the significance of each PCR result can only be achieved by adding a positive control to each single reaction The QIAGEN OneStep Ahead RT-PCR kit’s solution: • Duplex PCR optimized • Enable co-amplification of an internal positive control with every single reaction PCR1 PCR2 PCR3 With positive control PCR1: Shows both bands = PCR worked and TOI is present PCR2: Shows only control band = PCR worked but TOI is absent PCR3: Shows no band = PCR did not work > repeat experiment Without positive control PCR1: Shows band = TOI PCR2: No band means ? PCR3: No band means ? TOI = Target of interest ? ? ! ! 400 bp = TOI 300 bp = internal control PCR1 PCR2 PCR3 One Step Ahead for Your RT-PCR
Sample to Insight Eight pitfalls and one solution 1. Make sure that both assays work in a singleplex reaction 2. Don‘t combine assays with amplicon lengths > 1000 bp 3. Ideally, assays don‘t differ in length by more than 200 bp 4. If one band (i.e. assay) is clearly weaker than the other, increase annealing and extension step from 10 seconds to 20 seconds 5. If extended cycling times still don‘t give satisfying results, reduce the primer concentration for the “stronger“ assay by increments of 0.1 µM down to 0.1 µM, keeping the primer concentration for the “weaker“ assay at 0.5 µM Recommendations and optimization strategies 22One Step Ahead for Your RT-PCR
Sample to Insight 0,1 µM - Eight pitfalls and one solution Effect of optimization strategies Effect of cycling time extension Annealing/extension: 10 s Note: both targets (ACTB/VCL) differ by factor >100x in mRNA abundancy M M Effect of primer concentration NTC NTC - 0,5 µM - 0,5 µM 0,5 µM - Note: both targets (PPIA/CTNNA1) differ by >250 bp in amplicon length Annealing/extension: 20 s 23One Step Ahead for Your RT-PCR
Sample to Insight Eight pitfalls and one solution 24 Pitfall eight:Target RNA concentration threshold • Control experiments are necessary to tell whether the absence of a PCR product can be interpreted as a negative result or an unsuccessful PCR • Adding positive control to each single reaction providesfull proof about the significance of each PCR result The QIAGEN OneStep Ahead RT-PCRkit’s solution: • Chemistry optimized for highest sensitivity • 2.5x master mix allows for input of more sample RNA Indicated amounts of HeLa total RNA (in pg) w ere used as template for amplification of GAPDH (831bp) and ACTB (295bp) in duplicate, according to the suppliers’ instructions. Green arrow s indicate specific product, red arrow s indicate primer‐dimers. Analysis w as performed using the QIAxcel. 24One Step Ahead for Your RT-PCR
Sample to Insight How to use the kit Kit components Ultrapure water OneStep Ahead Master Mix Template Tracer RT-Mix Master Mix Tracer Q-solution 26One Step Ahead for Your RT-PCR
Sample to Insight How to use the kit 27 Reactionsetup for one-stepRT-PCR 27One Step Ahead for Your RT-PCR
Sample to Insight How to use the kit Ultrafast standard protocol for amplicons <1kbp From samples to insight in just 60 mins! Fastest results Reverse transcription step: OmniScript and SensiScript RTs are activated and reverse transcription takes place. The reaction temperature may be increased up to 55°C, if satisfactory results are not obtained at 50°C. Initial PCR heat activation This heating step activates DNA Polymerase blend, inactivates Omniscript® and Sensiscript® Reverse Transcriptases, and denatures the cDNA template. 10 min @50°C 5 min @95°C 10s Denaturation Annealing Extension Final extension RT PCR 40 cycles The optimal cycle number depends on the amount of template RNA and the abundance of the target transcript. 10s 10s 2 min 28One Step Ahead for Your RT-PCR
Sample to Insight How to use the kit Protocol for amplicons 1-4 kbp Fastest results Reverse transcription step: OmniScript and SensiScript RTs are activated and reverse transcription takes place. Initial PCR heat activation This heating step activates DNA Polymerase blend, inactivates Omniscript® and Sensiscript® Reverse Transcriptases, and denatures the cDNA template. 15 min @45°C 5 min @95°C 15s Denaturation Annealing Extension (Allow 1min per kbp amplicon size) Final extension RT PCR 40 cycles The optimal cycle number depends on the amount of template RNA and the abundance of the target transcript. 15s 1-4min 5 min 29One Step Ahead for Your RT-PCR
Sample to Insight How to use the kit 30 Comparison of cycling protocols Supplier QIAGEN Competitor A Competitor B Protocol RT 10 min / 50°C 30 min / 50°C 30 min / 55°C Taq activiation / template denat. 5 min /95°C 2 min /94°C 2 min /94°C Denaturation 10 sec / 95°C 30 sec / 94°C 15 sec / 94°C Annealing 10 sec / 55°C 30 sec / 60°C 30 sec / 60°C Extension 10 sec / 72°C 60 sec / 72°C 60 sec / 68°C Total runtime (incl. ramping) 60 min 137 min 127 min Our new kit is more than twice as fast as the competitors One Step Ahead for Your RT-PCR
Sample to Insight How to use the kit Primer Design, Concentration and Storage Standard RT-PCR primers Prerequisites for successful one-step RT-PCR include the design of optimal primer pairs, the use of appropriate primer concentrations and the correct storage of primer solutions. The QIAGEN OneStep Ahead RT- PCR Kit is designed to be used with gene-specific primers only. The use of random oligomers or oligo-dT primers is not recommended since this will result in the amplification of nonspecific products. 31One Step Ahead for Your RT-PCR
Sample to Insight Recommendations 33 Complete solution for RNAanalysis One Step Ahead for Your RT-PCR
Sample to Insight Recommendations • Enables fully automated DNA and RNAanalysis • Ready-to-rungel cartridges • Fast processing: 12 samples in 3 – 10 min • Up to 96 samples per run (unattended processingof up to twenty 96-well plates possible) • Sample input amounts < 0.1 µl • Detectionlimit of 0.1 ng/µl • High resolution of 3 – 5 bp up to 500 bp • Digital data output 34 Data analysis – QIAxcel One Step Ahead for Your RT-PCR
Sample to Insight Recommendations QIAGEN OneStep Ahead RT-PCR Kits https://www.QIAGEN.com/de/shop/pcr/end-point-pcr- enzymes-and-kits/one-step-rt-pcr/QIAGEN-onestep- ahead-rt-pcr-kit 35One Step Ahead for Your RT-PCR
Sample to Insight Recommendations QIAGEN OneStep Ahead RT-PCR Kits Sampling campaign Order your sample 50x Kit today One Step Ahead for Your RT-PCR
Sample to Insight Summary 38One Step Ahead for Your RT-PCR
Sample to Insight Summary QIAGEN OneStep Ahead RT-PCR Kit provides: • Convenient room temperature set up also enables use in automated/robotic workflows • Significantly shorter cycling safes time • Highest safety and piece of mind due to built in color change pipetting control • Highest template RNA protection due to built in RNase Inhibitor • Higher sequence accuracy and ability to amplify longer amplicons up to 4kb • Run internal positive control in the same reaction • Twice the amount of reactions per kit due to reduction of reaction volume • Convenient 2.5x master mix for easy calculation One Step Ahead for Your RT-PCR
Sample to Insight 40 Contact QIAGEN Call: 1-800-426-8157 (NA) +49-2103-29-12400 (EU) Dr. Bernd Willems: bernd.willems@QIAGEN.com Questions? Thank You for Attending One Step Ahead for Your RT-PCR

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