At the heart of every successful discovery lie the seeds of innovation. At QIAGEN, we are constantly developing new methods that allow researchers to gain forward momentum with their research. Whether you’re studying gene expression or performing viral RNA analysis, the success of your experiment depends on the ability to analyze your sample with the highest standards of sensitivity and specificity so that you can have confidence in your data. To help you generate valuable insights from gene expression profiling and viral RNA analysis, we introduce the brand new QIAGEN OneStep Ahead RT-PCR Kit – the first hot start reverse transcriptase kit on the market. Continuing the success story of its first-generation predecessor (QIAGEN OneStep RT-PCR Kit), the QIAGEN OneStep Ahead RT-PCR Kit is equipped with compelling new features that afford maximum convenience and ease of use, while delivering unmatched sensitivity and specificity. With a total reaction time of 1 hour, higher sequence accuracy and the ability to amplify amplicons of up to 4 kb without tedious optimization, you can get one step closer to publishing your findings with this new solution. For increased convenience, the kit comes in an all-in-one tube format along with a built-in pipetting control. Stay one step ahead of your peers and make significant advances in your research with the QIAGEN OneStep Ahead RT-PCR Kit! In this slidedeck, we introduce the new kit in detail and discuss its features and benefits.
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One Step Ahead for Your RT-PCR
1. Sample to Insight
One Step Ahead for Your RT-PCR
Dr. Bernd Willems, Global Product Manager
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2. 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.
6. Sample to Insight
Introduction
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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
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Eight pitfalls and one solution
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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
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Eight pitfalls and one solution
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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
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Eight pitfalls and one solution
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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
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Eight pitfalls and one solution
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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
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Eight pitfalls and one solution
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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
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Eight pitfalls and one solution
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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
14. Sample to Insight
Eight pitfalls and one solution
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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
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Eight pitfalls and one solution
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Pitfall two:Nonspecific amplification during reaction setup (cont’d)
One Step Ahead for Your RT-PCR
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Eight pitfalls and one solution
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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
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Eight pitfalls and one solution
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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
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Eight pitfalls and one solution
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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
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Eight pitfalls and one solution
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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
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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
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Eight pitfalls and one solution
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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
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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
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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
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Eight pitfalls and one solution
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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
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How to use the kit
Kit components
Ultrapure
water
OneStep Ahead
Master Mix
Template Tracer RT-Mix
Master Mix
Tracer
Q-solution
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How to use the kit
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Reactionsetup for one-stepRT-PCR
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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
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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
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How to use the kit
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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
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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.
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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
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Data analysis – QIAxcel
One Step Ahead for Your RT-PCR
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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
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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
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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