01%% 2'33-#%3$'%4,5.5%1#$3

!"#$%&'()*(+,-.'%/&01%%2'33"-#%3$'%4,5".5%1"#$3

Michael Tavaria, PhD

Scientific Applications Support Manager

Australasia

MHTP,

May, 2013

2 | Life Technologies Proprietary & Confidential | 8/05/2013

AgendaExperimental Design and the MIQE Guidelines

Sample Preparation

cDNA Synthesis

Assay design

Sample RNA Purification

Reverse Transcription qPCR



Sample Preparation

cDNA Synthesis

Assay design




Experimental Design

Samples

Treatment

BiologicalReplicates

Reference Genes

Controls

TechnicalReplicates

Sample Prep QPCR

Assays

RTase

Statistics

Analysis


The MIQE Guidelines

Why MIQE?Large number of Q-PCR publicationsDiverse reagents, protocols, analysis methodsLack of experimental detailsLack of consensus


The MIQE Guidelines


The MIQE Guidelines

Why MIQE?Large number of Q-PCR publicationsDiverse reagents, protocols, analysis methodsLack of experimental detailsLack of consensus

MIQE Goals:Better reliability and reproducibilityBetter transparencyBetter experimental practicesBetter ability to properly review submitted data Easier evaluation of published data



Sample Preparation

cDNA Synthesis

Assay design




Sample Preparation

Recovery

Extraction chemistry Integrity

Purity


Sample Preparation choices, choices ....


A complete solution from Sample Prep to Real-Time PCR from cultured cells.

Eliminates the need for RNA purificationMaximum sensitivity

Three easy steps: Lysis (including DNase treatment), Reverse Transcription and Real Time PCR

Rapid workflow<3 hours to results

TaqMan® Cells-to-Rapid, Easy, Complete QPCR Workflow


Cells-to-Ct - Rapid, Easy, Complete QPCR Workflow

10-100,000 cells<3 hrs total


Cells-to-



Sample Preparation

cDNA Synthesis

Assay design



25 | Life Technologies Proprietary & Confidential | 8/05/2013| Life Technologies Proprietary & Confidential |

cDNA synthesis primers:Oligo(dT)

Full length cloning

Gene Specific

Most accurate

Least flexible

Random Primer

Even distribution

Most flexible

Most common

Reverse Transcription

26 | Life Technologies Proprietary & Confidential | 8/05/2013| Life Technologies Proprietary & Confidential |

Reverse Transcription2-step:

Most flexible

More steps

Allows archiving

Most economical

1-step:Most convenient

Most sensitive

Most expensive

Least flexible


VILO = Variable Input Linear OutputExcellent linearity Ideal when input of RNA varies

> 1pg to 2.5ug of total RNA > no bias due to RNA input amounts

Ideal when abundance of gene varies> obtain the same relative representation in your cDNA

qPCR template, regardless of gene abundance

SuperScript III based with proprietary helper protein42°C working temperatureReduced inhibitionincreased yields

Superscript III® VILOReverse Transcription



Sample Preparation

cDNA Synthesis

Assay design




QPCR Assay Design - Chemistries


QPCR Assay Design - ChemistriesSyBr

Cheapest/entry levelCustom design (end-user responsibility for design)Pre-designed (primer pairs available from several sources BUT what quality of bioinformatics)Testing and validation required to ensure efficiency and specificityRe-testing and validation required Ongoing requirement for melt curves

TaqManMost specificMost sensitiveAbility to multiplexAccess to huge inventory of pre-designed guaranteed assaysCustom design using universal design guidelinesCost effective

Confidence

Confidence ?????


Primer:Primers match target and are in the correct 5' 3' orientationNo runs of more than 3 consecutive Gs in either primerNo more than 2 GCs in the last 5 nucleotides at the 3' endGC Content (20 80 %)Tm of 58 60°C

TaqMan Probe:Probe matches target and is in the correct 5' 3' orientationNo runs of more than 3 consecutive GsNo G on 5' endProbe selected from the strand with more Cs than GsGC Content (20 80 %)Tm of 68 70°C for GEx and 65 67°C for SNP ADFor SNP AD, polymorphism is near centre of probe

Amplicon lengthBetween 50-150bp

Universal Assay Design Guidelines


!"#$%&'&"(#)**+,-#*.*/0#12&'"310&#

!"'*4&3&*(5*4&356&7-89:01;<=+>

!"2$?@53A?@53B583/"20&#

%&(20&C&6%,D

+&E8&31&7%&F+&E>

TaqExpress

%83*G"E-0'&=&#0H3*-206&2#)

-259&#

G2"3#120'(I<*

7B:"#(>

4&356&I<*

7B:"#(>

!"#$%&%'( I<A+&:&1(*(5'*#15203H*

"##"J#

!"#$%&'&"(#)**+,-#*.*/0#12&'"310&#

D::*K0HL*+15203H*D##"J#*DC"0:"9:&*53*M&9#0(&

Currently over 1.2 million GEx & 4 million SNP Assays!

TaqMan® Assays Assay Design Process


Over 1.2 million assaysFlexible formats starting from <$10019 model organismsUniversal thermal cycling conditionsGuaranteed assay performance

TaqMan® Gene Expression Assays


TaqMan® Assays - Scientific BenefitsUniversal reaction conditions

All Gene Expression assays amplify using the same cycling and reaction conditions. Any combination of targets can be examined on the same plate

> Increased flexibility for evolving projects> Any endogenous control(s) can be selected> More data made available quickly and effectively

Guaranteed assay performance

Highly Specific

Robust

Multiplexing capability

Advanced Bioinformatics used in Assay Design


TaqMan® Assays Broad Product Range

mRNA Translocations

CNV

Bacteria Virus

miRNA

SNP Protein

Expression

Not just

mRNA

Somatic Mutations


SummaryExperimental Design and the MIQE Guidelines

Sample Preparation

cDNA Synthesis

Assay design



Every step of this process is important


QPCR Methods AQ vs RQAbsolute Quantification

Requires standards measured in units eg copies/ulMost commonly used for clinical/diagnostic tests

Relative QuantificationMost commonly used for gene expression Two methods:

> Relative Standard Curve (RSC)Uses a standard with arbitrary units (eg a sample)Can be used without knowledge of reaction efficienciesStandard curve required with every run

> Comparative ( Ct)A standard curve is only required initially to test reaction efficiencyExperiments do not require standard curvesAdjustments can be made for known reaction efficienciesMost efficient, uses least reactions


QPCR Methods - NormalisationRelative Quantification relative to what?

Required to adjust for different amounts of sampleCells ---- RNA ----- cDNA

Endogenous Control/Reference Gene> Traditional method> Need to identify consistent expressed gene> Can sometimes be difficult> Use multiple endogenous controls?

Global Normalisation> Works with large numbers of targets (>100)> Mean of all Ct values is more reliable normaliser

Ct


QPCR Methods - NormalisationCt values are arbitraryCt values are logarithmic

How do we translate Ct values to Relative Quantities?Ct is a measure of target gene expression relative to normaliser

> in one sampleCt is a measure of target gene expression relative to normaliser > across samples

Need to choose one sample as the calibrator or reference sample

Need to convert these logarithmic values to linear values2^ Ct (for 100% reaction efficiencies, the 2 is valid)


QPCR Methods Amplification Plots

GeometricAmplification

Threshold

Baseline


GeometricAmplification

Threshold 1

Baseline 2

Threshold 2

Baseline 1

QPCR Methods Amplification Plots


Example RQ data


Example RQ data

Brain is the referencesample = 1

Upregulation

Downregulation


Standard Curve: Test Assay PerformanceBest way to test assay performance

Create dilution series over wide dynamic range (4-6 10-fold dilutions)

High correlation coefficient

Slope close to -3.3 suggests assay is working optimally (100% reaction efficiency).


Troubleshooting - Multicomponent plot

Passive reference(ROX) - Flat

Normal amplification


SyBr Green What & Why Melt Curves?These are post-PCR stages that assess the melting temperature of the product(s) amplified during our reactions.

Ideally, we want to see one clean peak at the expected Tm in each well, as this suggests only our target amplified.


SyBr Green What & Why Melt Curves?


Troubleshooting Questions to AskPlate Set up

Most common cause of issues> Correct labelling of detectors/targets> Correct assignment of wells> Leave blank wells empty> Check Passive Reference set appropriately

Run MethodCorrect thermal cycling porotocolTaq activation step criticalData collection at appropriate step

AnalysisAuto Ct & BaselineTry Manual Baseline


Troubleshooting Online help


Troubleshooting Tech Support1800 636 327, Option 3

[email protected]

Thank You


Appendix Reaction Efficiency Calculation

A 100% efficient reaction will amplify a target 10-fold in 3.32 cycles(ie. 2 3.32 = 10 = 100%)

To calculate reaction efficiency:E = [10(-1/slope) 1] x 100

For comparative ( Ct) experiments to be valid, the reaction efficiencies of targets and endogenous control should be +/- 10% of each other.

01%% 2'33-#%3$'%4,5.5%1#$3

Documents