Ampliqon seminar 29 , 30 Nov 2015 by Viragene Akam Co

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Perfect Results a matter of knowledge

Mark N. Møller Molecular Biologist and developer of RealQ Plus

Master Mix

IRAN Distributor , Viragene Akam Co . Tel : (+9821) 88611552-3, 88062042-3

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Ampliqon Real-Time Mixes

December 7, 2015 Mark N. Møller 2

RealQ Plus Green 2x Master Mix • A323402 - Without ROX • A324402 - Low ROX • A325402 - High ROX

RealQ Plus Probe 2x Master Mix • A313402 - Without ROX • A314402 - Low ROX • A315402 - High ROX


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Workflow

Sample

RNA isolation/ purification

DNA isolation/ purification

cDNA synthesis

qPCR Data analysis



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Disposition

Definitions and Concepts Real-time PCR chemistry Optimizing and analyzing data

o Primer optimization and PCR protocol o Amplification Plot o Standard Curves o Multi Component Plot o Melt Curve

Example Conclusions December 7, 2015 Mark N. Møller 4


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Definitions and Concepts

qPCR = Quantitative real-time PCR

Quantification not quantitation

Cq = Quantification cycle (Ct, Cp and TOP)

Reference genes = Genes used for normalization


The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments. Clinical chemistry 55:4, 611-622 (2009). Stephen A. Bustin et al.


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Analytical Sensitivity:

”Minimum number of copies in a sample that can be measured accurately with an assay”.




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Analytical Specificity:

”the qPCR assay detecting the appropriate target sequence rather than other, nonspecific targets also present in a sample”.




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Repeatability:

”the precision and robustness of the assay with the same samples repeatedly analyzed in the same assay. It may be expressed as the SD for the Cq variance”




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Reproducibility:

”the variation in results between runs or between different laboratories and is typically expressed as the SD or CV of copy number or concentrations”



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Disposition



Examples Conclusions December 7, 2015 Mark N. Møller 10


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Real-time PCR Chemistry


RealQ Plus Green 2x Master Mix • A323402 - Without ROX • A324402 - Low ROX • A325402 - High ROX

RealQ Plus Probe 2x Master Mix • A313402 - Without ROX • A314402 - Low ROX • A315402 - High ROX


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Real-time PCR Chemistry SYBR Green

• ≥ 1000-fold increase • Fluorescent signal is proportional to the amount of dsDNA • Binds all dsDNA (unspecific)

”the qPCR assay detecting the appropriate target sequence rather than other, nonspecific targets also present in a sample”. - MIQE


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Real-time PCR Chemistry Hydrolysis Probe

• TaqMan is a hydrolysis probe • One reporter signal One amplified target • Fluorescence when intended target is amplified (Specific)

”the qPCR assay detecting the appropriate target sequence rather than other, nonspecific targets also present in a sample”. - MIQE


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Real-time PCR Chemistry ROX Passive Reference Dye


Intended to normalize for non-PCR related fluorescence variations ROX can correct for: - Air bubbles - Fluorescence variations because of concentration and volumes (limited) - Fluorescence variations beacuse of sample effects (limited)

ROX can identify: - Evaporation - Poor mixing - Some pipetting errors

Excitation spectrum Emission spectrum

585 nm 605 nm


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Real-time PCR Chemistry ROX Passive Reference Dye IRAN Distributor , Viragene Akam Co .

Tel : (+9821) 88611552-3, 88062042-3

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Disposition

Definitions and Concepts Real-time PCR chemistry principles Optimizing and analyzing data

• Primer optimization and PCR protocol • Amplification Plot • Standard Curves • Multi Component Plot • Melt Curve



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Optimizing and Analyzing Data Primer optimization and PCR protocol

Primer design affects specificity, efficiency and accuracy. • Find target sequence (NCBI) • Upload correct sequence to primer software

• Primer 3 • http://primers.gene-quantification.info/ • PrimerExpress (ABI)

• Important parameters • Melting temperature (TM) • GC-content • Target length (50-150 bp) • Self priming

• Primer concentration (100-500 nM)



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Initial heating

15 min.

Cycling

15 - 30 sec.

30 sec.

30 sec.

Melt Curve

15 sec.

15 sec.

1 min.


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40 °C 50 °C 45 °C 55 °C 60 °C

Mismatch vs. perfect match

”Although primer optimization has become unfashionable, it is clear that poor annealing optimization has a large effect on assay quality” - MIQE


A T T C G A A T C

A T A A T C G A C A A T T G A C G A A T T A A

A T C G T A A T T T T C C C G G G G T T A A T T T T C C C A A A A T T T T C C A A G G


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Disposition


o Primer concentration and PCR protocol o Amplification Plot o Standard Curves o Multi Component Plot o Melt Curve



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Optimizing and Analyzing Data The amplification plot


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0,5

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1,5

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2,5

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3,5

4

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40

ΔR

n

Cycle

Lag Phase

Plateau Phase

Linear Phase

Log Phase

Linear View vs. Log View

0,01

0,1

1

10

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40

ΔR

n

Cycle

Lag Phase

Log Phase

Linear Phase

Plateau Phase

Log phase is the most important phase!


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Optimizing and Analyzing Data Lag Phase - Baseline


Rn dRn

• Spread lag phase • Different cross points

• Flat lag phase • One y-axis cross point in 0


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Too Low! Increase the end cycle

Too high! Decrease the end cycle and start cycle


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Baseline set correct!


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1

1,5

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3,5

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0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40

ΔR

n

Cycle

Lag Phase

Plateau Phase

Linear Phase

Log Phase (Exponential)


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Optimizing and Analyzing Data Linear and Plateau Phase


”After 30 cycles of PCR, well over 90 % of primers and nucleotides are not consumed

and once the plateau is reached, amplification cannot be restarted by adding

fresh reagents”

J. Hedman, doctoral thesis: ”DNA Analysis of PCR inhibitory forensic samples”, 2011.


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Optimizing and Analyzing Data Linear and Plateau Phase

Two main factors on linear and plateau phase: • Target product inhibition of Polymerase • Annealing competion: dsDNA self-annealing vs.

primer annealing


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How can we use the amplification plot to optimize, analyze and help us generate the perfect results?


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Optimizing and Analyzing Data Log Phase – Defining Cq


Second derivative maximum method:

Cq - value Second derivative of amplification plot

Amplification Plot


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1,5

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3,5

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0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40

ΔR

n

Cycle

Perfect

Fluorescence threshold – Linear View

Cq - value

Too high

Too Low


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0,01

0,1

1

10

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40

ΔR

n

Cycle



Perfect

Fluorescence threshold – Log View

Cq - value

Too high

Too Low


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Let’s look at it


Steep

Flat (Inhibitors)

Modified from J. Hedman, doctoral thesis: ”DNA Analysis of PCR inhibitory forensic samples”, 2011.


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Check list for perfect results Amplification Plot


Sigmoidal curves with four phases

Steep sigmoidal curve

Standard deviation ≤ 0.2 between replicates

Negative controls (NTC) within limits (≥ 40 cycles)

Consistent spacing (Standard curves)

Checklist:


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Break

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Disposition





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Optimizing and Analyzing Data Standard curve


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-1,5 -1 -0,5 0 0,5 1 1,5 2 2,5

Cq

Log ng DNA

Standard Curve

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1

2

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10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42

ΔR

n

Cycle

Amplification Plot

Efficiency = How efficient the polymerase amplifies the target. 1 cycle with 1 DNA doubling = 100 % efficiency


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”Amplification efficiency should be determined from the slope of the log-linear portion of the calibration curve… The dynamic range should

cover at least 3 orders of magnitude and ideally should extend to 5 or 6 log10 concentrations.”

S. Bustin et al.: ”The MIQE guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments”, 2009.


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PCR efficiency = 10−1/𝑠𝑙𝑜𝑝𝑒 − 1

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-1,5 -1 -0,5 0 0,5 1 1,5 2 2,5

Cq

Log ng DNA

Standard Curve

R2 = Statistical measure of how close the

data are fitted to the regression line (≥ 0.98)


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For a precise standard curve, it important to: - Prepare the dilution series from the previous tube (change filter tips in

between) - Dip the tip as little as possible and pipet carefully and use low retention

tips - Calibrate pipets and use sufficient volumes - Have at least three replicates - Mix thoroughly, but do not vortex nor pipet up and down when mixing

doublestranded DNA - Use same DNA as target


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How can we use the standard curve to optimize, analyze and help us generate the perfect results?


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Too low Efficiency Too high efficiency


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0 0,5 1 1,5 2 2,5 3

Cq

Log ng DNA

y = -3.7411x + 29.142 R2 = 0.9781

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0 0,5 1 1,5 2 2,5 3

Cq

Log ng DNA

y = -3.0767x + 28.277 R2 = 0.964

= 10−1/−3.7411 − 1

= 0.851


= 10−1/−3.0767 − 1

= 1.114

= 111.4% = 85.1 %

90 – 110 % (-3.6 > Efficiency > -3.1)

100 % = -3.322


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• Too low efficiency = reached sensitivity limit

• Too low efficiency = lack of annealing temperature or primer concentration optimization

• Too low efficiency = lack of primer sequence optimization

• Too high efficiency = Inhibitors and lack of sample concentration optimization

• Too high efficiency = Too high primer concentration

• Too high efficiency = Unspecific amplification

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Sensitivity: ”Minimum number of copies in a sample that can be measured accurately with an assay”.


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”PCR efficiency is particularly important when reporting mRNA concentrations for target genes relative to those for reference genes. The ΔΔCq method is one of the most popular means of determining

differences in concentrations between samples and is based on normalization with a single reference gene…The 2 genes must be amplified with comparable efficiencies for this comparison to be

accurate.” - MIQE

Relative quantification


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Check list for perfect results Standard Curve


Efficiency between 90-110 %

R2 value ≥ 0.98

Efficiency is not falsely high

No banana shaped standard curve

Checklist:


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Disposition





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Optimizing and Analyzing Data Multi component view


ROX

SYBR

Amplification Plot

Melt Curve

Fluorescence raw data


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Optimizing and Analyzing Data Multi Component View


How can we use the multi component view to optimize, analyze and help us generate the perfect results?


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Optimizing and Analyzing Data Multi component view


Low SYBR signal: • Failing bulb • Mix expiration High SYBR signal • Can inhibit PCR ROX signal inconsistency: • Evaporation • Poor mixing • Some pipetting errors

Cq Values: • Manipulative with SYBR • Manipulative with ROX

ROX

SYBR

Cq difference


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Check list for perfect results Multi Component View


Reporter within range and at the desired levels

ROX passive reference dye signal is unchanged

ROX level correct and help maintain low standard deviation

Checklist:


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Disposition





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Optimizing and Analyzing Data Melt Curve

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Intensity

Temperature TM

50 %


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50 °C 95 °C

Flu

ore

scen

ce In

ten

sity


Temperature


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dI/dT

Temperature

TM

Mark N. Møller

Temperature

- dI/dT

TM


Intensity

Temperature TM

50 %


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Target Melt point influenced by: • DNA amount • pH • Buffer composition


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Different buffers can give different TM

=> Optimize annealing temperature to your specific buffer


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How can we use the melt curve to optimize, analyze and help us generate the perfect results?


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Unspecific product

Target

• Redesign primers • Optimize annealing temperature • Optimize primer concentration

Target

• Have separate work spaces • Setup • PCR run • Opening plates

+ NTC (contamination)

Unspecific product


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Primer dimers

Target

• Redesign primers • Optimize annealing temperature • Hot Start enzyme

Primer dimer formation


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Check list for perfect results Melt Curve


Clear and distinguished peak at intended temperature

No primer dimers

Negative NTC’s

No unspecific amplification

Checklist:


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Break

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Disposition





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Example Absolute Quantification


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1

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ΔR

n

Cycle

Amplification Plot

SD = 0.07


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-1,5 -1 -0,5 0 0,5 1 1,5 2 2,5

Ct

Log ng DNA

Standard Curve

Slope: -3.376 R2: 0.999


= 10−1/−3.376 − 1

= 0.978

= 97.8 %


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0

10000

20000

30000

40000

50000

60000

70000

80000

90000

100000

110000

120000

130000

60 65 70 75 80 85 90 95 100

Der

ivat

ive

Rep

ort

er (

-R')

Temperature (°C)

Melt Curve


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Disposition





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Conclusions

To get the best results it is important to know:

• How to set up and optimize your experiment

• How to analyze your results

• This can be done with Ampliqon RealQ Plus Master Mixes!



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Education

Ampliqon seminar 29 , 30 Nov 2015 by Viragene Akam Co