template quality and bioa - agilent · template quality and real-time qpcr influence of template...

Template Quality and Real-time QPCR

Influence of template quality on real-time QPCR results

Cathy Cutler Field Application Scientist

Our measure is your success.

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1.Experimental Design

2.Sample preparationand purification

cDNATotal RNA

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4.Reverse Transcription5.Real time QPCR6.Post-run Analysis

Steps towards successfulReal-time QPCR experiments

3. RNA/DNA quantification and quality

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Why Quality MattersRNA/DNA quality control (Quality of template):

RNA degrades naturally due to enzymatic or autocatalytic mechanisms:Any 5’ or 3’ biased design might fail or produce misleading resultsWrong priming strategy in the RT step can lead to misleading results

Knowing RNA/DNA quality allows to accommodate the design and set expectationsavoiding wrong interpretation of results

All quantifications rely on comparable template quality to be meaningfulQPCR assay validation (Quality of results):

The resolution of SYBR Green meltcurves is limited

Tm depends on dye/template ratio

SYBR Green is a non-saturating dye with non-uniform distribution along thedoublestrand

Verifying the size of PCR products is a recommended validation procedureResolution of slab gels again is limited!

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Sample PurificationSample purification greatly influences QPCR results:

Buffers from sample purification can interfere with downstreamRT or QPCR (eg. cell culture medium, etc.)

Co-purified salts influence primer/probe binding characteristics

Co-purified inhibitors can lead to failure of PCR(eg. Phenol, poly-saccharides, heme, lipids, heparin etc.)

Timing and way of purification can impact template quality

Optimizing sample purification enhancesQPCR efficiency and success

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Sample Purification RNA

1 human cell contains: 10-5 µg RNA

80-85% rRNA (5-, 5,8-, 18-, & 28S)15-20% low-molecular weight abundant RNA1-5% mRNA

RNA Isolation not so different from DNA isolationProblem is not the instability of RNA, but the stability of RNases

mRNA vs. Total RNA purification:- Not all mRNAs have a poly-A tail- Length of poly-A tail varies

Affinity of mRNAs to oligo-dT based purification systems varies!mRNA purification might introduce a bias

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Sample PurificationChallenges with FFPE samples

FreshFrozen

FFPE

Fixation alters the nucelic acids:Crosslinking between protein and nucleic acid fragmentation

Purification methods time consuming (several hours – over night)

RNA can be highly degraded:Don‘t use oligo-dT for RT primingUse of very small (≤100 bp) amplicons

Frozen tissue

FFPE tissue

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Sample PurificationChallenges with FFPE samples

Absolutely FFPE16h 3h

Competitor1.5h/16h

Phenol

NTC

Phenol47.416h

16.316hAbsolutly RNA FFPE

16.21.5h

32.216hCompetitor

27.41.5h2

Phenol8.316h

5.216hAbsolutly RNA

FFPE

4.83h

9.216hCompetitor

8.21.5h1

Methodμg RNAProtKSample

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Why DNA Quality matters

Assayperformance

andsuccess

Small DNA fragmentscompete with primers:Unspecific amplification,

Reduced population of DNAwith full length of amplicon:Underestimation of quantity

Alterations of bases:Reduced affinity of primersand probes

DNA degradation in preserved biological tissue, forensic samples orsamples commonly used in pathogen detection can negatively impactassay performance and produce misleading results

Competition by abortiveamplicons:Loss of sensitivity or inhibition

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Comparative Quantification

Gene of Interest

Given two samples: What is the difference in gene expression?

Reference gene(Normalizing assay)

Calibrator(Control) Unknown 1 Unknown 2 …..

±x times

Ct Ct Ct

±x times

Expression changeof GOI relative to Calibrator

Expression changeof Normalizer relativeto Calibrator

GOI Normalized changerelative to Calibrator

Ct Ct Ct

Assumes Calibrator and Unknown samples are comparable!

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Why RNA quality matters –Intact RNA

GOI

REF

ΔΔ

CtF

old

Cha

nge

of G

OI

2 21 1

2 2 2 2

ΔC

tfor

GO

I and

RE

F

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Why RNA Quality Matters –Degraded RNA

GOI

REF

ΔC

tfor

GO

I and

RE

F

ΔΔ

CtF

old

Cha

nge

of G

OI

2 20.5 1

4 2 4 2

Example: 50% of all REF RNAs are degraded from 5‘ end:

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Experimental workflowRNA extraction

Nucleic acid quantificationand QC

Reverse Transcription

QPCR Assay validationQPCR 5‘ and 3‘ assays

Extraction from 5x106

HEK cells usingAbsolutely RNA® mini

RT from 1 µg of totalRNA usingAffinityScript™

Quantification of 1 µlsample on NanodropQC on Bioanalyzer:RNA 6000 nano kit

Analysis of QPCR products onBioanalyzer: DNA 1000 kit

RNA degradation @ 70°C

Mx3005P®

Brilliant® II SYBR® Green

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Experimental DesignLook at the influence of RNA quality on interpretation of QPCR results

Look at endogenous transcripts of varying expression levelUse 5’ and 3’specific assaysUse oligo dT and random hexamersIntentional degradation of RNA

Illustrate the value of the BioAnalyzer in evaluation of PCR products post reaction

Interpretation of non-specific products

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High quality total RNA(RIN 8.8)

mar

ker

Partially degraded total RNA(RIN 3.7)

Assesment of RNA IntegrityRNA 6000 Nano LabChip kit

mar

ker

Typical first QC step during cDNA or cRNA sample prep for QPCR

2100 bioanalyzer: single lane gel-like image

2100 bioanalyzer: electropherogram

18S28S

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Assessment of RNA Integrity

Intact RNA: RIN 10

Partially degraded RNA: RIN 5

Strongly Degraded RNA: RIN 3

18S

28S

Fluo

resc

ence

0123456789

18S

28S

Fluo

resc

ence

05

1015202530354045

Fluo

resc

ence

Time (seconds)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

19 24 29 34 39 44 49 54 59 64 69

*Fleige, S.; Pfaffl, M (2006) Molecular Aspects of Medicine 27:126-139

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Assesment of RNA IntegrityRNA Integrity Number (RIN)

RIN available for eukaryotic RNA only !!

What the RIN can do:Obtain an assessment of RNA integrity.Directly compare RNA samples Ensure repeatability of experiments

What it CANNOT do:Predict the outcome of an experiment if no prior validation was done

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Assay: RNA Integrity

Total RNA was purified from 5x106 HEK cells and analyzedon the 2100 bioanalyzer.

The value of 10 obtained for the RIN shows the very high integrity of thepurified sample.

18S

28S

mar

ker

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Assay: Design

GAPDH: Glyceraldehyde-3-phosphate dehydrogenase (Chr. 12, 9 exons)

HPRT1: Hypoxanthine-guanine phosphoribosyl transferase (Chr. X, 9 exons)

YWHAZ: Protein kinase C inhibitor protein 1 (Chr. 8, 6 exons)intron 2-3: 23.6 kb

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NTC

NTC 21 + 51 bp

Assay: Validation BioAnalyzer Discordant results with SYBR Melt

Size: 120 bp

All assays had clear noRT controls both for QPCR Reference RNA and HEKtotal RNA no gDNA present (YWHAZ 3‘ assay is in one exon!)

One NTC was positive for the HPRT1 5‘ assay (167 bp):

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Assay: ValidationBeyond Melting Curve Analysis BioAnalyzer

Why? SYBR Green is a non-saturating dyeMelting temperatures of products

can be misleading based upon length and sequence BioAnalyzer analysis will give

accurate high resolution information about what is being made

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oligo-dT

random

GAPDH 5‘ assay: Expected size 118 bp

Size: 110 bp

HPRT1 3‘ assay: Expected size 114 bpoligo-dT

random

Size: 102 bp

Assay: Validation BioAnalyzer Concordant results with SYBR Melt

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Assay: ValidationReal-time quantitative PCR

HPRT1 5‘ assay: Expected size 130 bp HPRT1 3‘ assay: Expected size 114 bp

oligo-dT

random

oligo-dT

random

Size: 120 bp Size: 102 bp

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Assay: ValidationReal-time quantitative PCR

YWHAZ 5‘ assay: Expected size 142 bp YWHAZ 3‘ assay: Expected size 128 bp

oligo-dT

random

oligo-dT

random

Size: 125 bp Size: 116 bp

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RNA Quality and ExpressionLevels

RIN 8.90 min

A RIN 6.530 min

B

RIN 4.645 min

C RIN 2.375 min

D

-14

-12

-10

-8

-6

-4

-2

0RIN 8.9 RIN 6.5 RIN 4.6 RIN 2.3

GAPDH 5' assayGAPDH 3' assay

-10-9-8-7-6-5-4-3-2-10

RIN 8.9 RIN 6.5 RIN 4.6 RIN 2.3

HPRT1 5' assayHPRT1 3' assay

GAPDH

HPRT1

YWHAZ

-6

-5

-4

-3

-2

-1

0RIN 8.9 RIN 6.5 RIN 4.6 RIN 2.3

YWHAZ 5' assay

YWHAZ 3' assay

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SummaryDNA/RNA quality can have a dramatic effect on QPCR results

Performing quantification assumes comparable templatequality

Extent of effect is sequence/gene dependent

For successful QPCR it is advisable to optimize samplepreparation methods to achieve highest template quality possible

Adjust design of amplicon (position/size) based on sample quality

The Bioanalyzer allows easy assessment of RNA qualityand facilitates QPCR assay validation

DNA fragment analysis on Bioanalyzer more sensitive as SYBR Green meltcurve

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Additional Information

http://www.chem.agilent.com/scripts/LiteraturePDF.asp?iWHID=54545

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How can the BioAnalyzer improve QPCR gene How can the BioAnalyzer improve QPCR gene expression Analysis?expression Analysis?

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Gene Expression WorkflowRNA extraction

Nucleic acid quantificationand QC

Reverse Transcription

QPCR Assay validation

QPCR

Absolutely RNA®

AffinityScript™

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Bioanalyzer Lab-on-a-Chip General Features and Benefits

Smaller - Faster - Smarterfrom sample to digital data - quickly and reproducibly

Miniaturization (Scale)• small sample volumes• reduced reagent usage• reduced bench space

00:00:1500:00:1500:00:1500:00:1500:00:1500:00:1501:30:00

Automation• improved accuracy• improved precision• improved productivity

Miniaturization (Speed)• fast analysis

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Lab-on-a-Chip Technology An Overview

Based on Microfluidics

the movement of liquids through micro-fabricated structures by means of electrical fields or pressure/vacuum

small glass or plastic devices with micro-channels as experimental platform active control of fluids without moving parts on-chip

through miniature electrodes or pumpscontrolled by software scripts

emulation of conventional liquid pumps, valves, dispensers, reactors, separation systems, etc.capability of liquid transfer, separation, dilution, reactions and more

…holding the promise of greater functionality with significantly improved reliability!

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Run Gel

30 min-2 hours

Prep Gel

15 min

2 hours- 1 day

Scan / Analyze

30 min

Stain / Destain

30 min-8 hours

Prep Chip

15 min 40 min

Run Chip Analyzed - Archived data

25 min

Lab-on-a-Chip or Gel

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2100 Bioanalyzer HardwareBayonet Cartridge

Exchangeable cartridge for electrophoresis or flow cytometry assays

16 pin electrodes connected to HV-sources

Chip holder with heater plate

Optics for detection

Chip selector

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Chip for Molecular AssaysChip for Molecular AssaysAnalysis of DNA, RNA and proteins. Analysis of DNA, RNA and proteins.

• Chip accommodates sample wells, gel wells and a well for a standard (ladder)• 16 pin-electrodes in the electrode cartridge (standard equipment) are arranged such that they fit in the wells on the chip• on-chip gel electrophoresis

Gel wells

Ladder well

Sample wells

Gel wells

Ladder well

Separation channel and point of detection

DNA chip displayed as example.

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Principle of Electrodriven Flow

1. The sample moves electro-driven from the sample well through the micro-channels

2. The sample is electro-kinetically injected into the separa-tion channel

3. Sample components are electro-phoreticallyseparated

4. Components are detected by their fluorescence and translated into gel-like images (bands) and electrophe-rograms (peaks)

The micro-channels of the glass chip are filled with a sieving polymer and fluorescent dye

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Principle of Electrodriven Flow

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The Lab-on-a-Chip Approach

Active Control of Fluids without Moving Parts

Sample volumes 1 -4 µl

10 -12 samples depending on Assay

Separation, staining, detection of samples

Results in 5-30 minutes available

No extra waste removal needed

Disposable Chip, no crosscontamination

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RNA Applications

RNA QA/QC for Microarrays

Gene Expression

Genomic DNA

ContaminationRNA QA/QC for

qPCR

RNA QA/QC for mPCR

smallRNAQA/QC

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Protein ApplicationsAntibodyQA/QC

Protein Expression

Protein Purification

Food Analysis

ProteinQA/QC

Compliant Protein & AntibodyQA/QC

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DNA Applications

mtDNAScreening

qPCR validation, impurity check

mPCR validation, impurity check

Restriction Digest Analysis

Gene Expression

Oncology

Food Analysis

Clinical Research

ForensicTesting

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Cell Applications

ApoptosisAnnexin V

ApoptosisCaspase-3

Transfection EfficiencyGFP

Transfection EfficiencysiRNA

Transfection EfficiencyAntibody Staining

Protein Expression Monitoring

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Current AssaysIntrod

uctio

n

DNA Assays:• Sizing• Quantitation• PCR products, digests,

larger DNA fragments• 12 samples in 30 min.

DNA1000 DNA7500 DNA12000

RNA Assays:Quantitation (Sizing in Small

RNA)total RNA, mRNApurity & integrity determination10 samples in 30 min.

6000 Nano 6000 Pico Small RNA

Cell Assays:

• Analysis of 6 samples • Two color detection• Analysis of protein expression

in cells

Flow Cytometry

Protein Assays:• Sizing

• Quantitation

• cell lysates, column fractions,

purified proteins, antibodies etc.

• 10 samples in 40 min.

P230 P80 P250

template quality and bioa - agilent · template quality and real-time qpcr influence of template...

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