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  • Influence of RNA matrix effect on qRT-PCR results- an overview

    Michael W. Pfaffl & Simone Fleige

    qPCR 2005, Freising-Weihenstephan, 5 7th September 2005

    Michael W. PfafflPhysiology - WeihenstephanCenter of Life and Food SciencesTechnical University of Munich Weihenstephaner Berg 385350 Freising-Weihenstephan; Germanymichael.pfaffl@wzw.tum.dewww.gene-quantification.info

    http://www.gene-quantification.info/

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    cycles

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    (line

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    Fluorescence history

    4 samples in quadruplicatesSYBR Green I performed in LightCycler 1.0

  • PCR inhibitors:Hemoglobin, Urea, Heparin

    Organic or phenolic compoundsGlycogen, Fatty acids, Ca2+

    Tissue matrix effects ( ?!?!? )Laboratory items, powder, etc.

    PCR enhancers:DMSO, Glycerol, BSA

    Formamide, PEG, TMANO, TMAC etc.Special commercial enhancers:

    Gene 32 protein, Perfect Match, Taq Extender, AccuPrime, E. Coli ss DNA binding

    real-time PCRefficiency & performance

    RNA / DNA degradation

    PCR reaction components

    DNA concentration

    sampling and tissue degradation

    unspecificPCR products

    hardware:PCR platform & cups

    lab management DNA dyes cycle conditionsPfaffl, qPCR 2004

  • Steps and variables of a successfulmRNA quantification using real-time RT-PCR (1)

    nucleic acidisolation

    samplepreparation

    RT = reversetranscription

    real-time PCRamplification

    Sampling method: Biopsy Fixed material Fresh blood Tissue storage Liquid Nitrogen RNA Later 1st extraction buffer=> native RNA

    Extraction method:total RNA vs. mRNA liquid-liquidcolumnsby RobotRNA integrity:

    Bioanalyzer, Experion Nano Drop various dyes

    RNA storage 80C

    Efficiency of RT: RT enzyme type RT temperature poly-T Primer Random-hexamers Specific primer Primer mixtures one-step RT-PCR two-step RT-PCR

    Efficiency & Specificity of real-time PCR:

    Primer design Primer specificity

    multi-species Primer mRNA abundance cDNA input Polymerase types & Mixtures Robot vs. Technician

    Pre-Analytical steps:

    tissuesample

    RNA cDNA PCR

  • Steps and variables of a successfulmRNA quantification using real-time RT-PCR (2)

    quantification strategy

    RT-PCR product

    software

    Detection method: SYBR Green I Probes: Beacons, Scorpions etc. Fit point method TaqMan fitting (10x SD) 2nd derivative maximum other models (Log. or Sig.) raw data vs.background correction other curve manipulations 2-step, 3-step or 4-step PCR

    Quantification strategy: absolute quantification

    which standard curve normalization with HKG

    relative quantification Total RNA, mass of tissue normalization with HKG normalization via an index of

    more (> 3) HKGs geNorm, REST, BestKeeper,

    qBASE, Normfinder, etc.

    BioInformatics: CP vs. molecules Normality of data (???) t-Test (?) ANOVA (on the ranks ?) SAS, SPSS, Excel, Sigma Stat Permutation test Randomization test (REST) .???

    detection

    statisticssuccess

    = biological

    meaningfulresults

    test

  • Determination the extraction efficiency

    total RNA extraction

    tissue sample:Liver, Spleen,

    Kidney, CaecumColon, Reticulum

    Spike with rec. RNA

    100 100,000 molecules per tube

    OD260

    RTreverse transcription

    qPCR

    CP dataanalysis

    standard curveusing rec. DNA

    10 100,000 molecules

  • Tissue extraction efficiency [ meansem ](3 tissue repeats with 4 different spikes, n =12)

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    Caecum Spleen Kidney Liver Colon Reticulum

    extr

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  • RT EfficiencyQiagen SYBR Green I qRT-PCR Kit, performed in LightCycler

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    1 2 3 4mean daily recovery of recRNA MultiStandard (100 - 10,000 molecules)

    overall mean = 58.517.5%

    RT

    effic

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    y [ m

    ean

    std.

    dev.

    ]

  • RT EfficiencyQiagen SYBR Green I qRT-PCR Kit, performed in LightCycler

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    100 1000 10000 100000spiked molecules recRNA MultiStandard per reaction setup

    overall mean = 61.317.4%

    RT

    effic

    ienc

    y [ m

    ean

    std.

    dev.

    ]

  • Stahlberg et al., Clin Chem. 50(9) 2004

  • Influence of total RNA quality, quantity and purity on qRT-PCR resultstotal RNA extracted bovine WBC analysed in Bioanalyzer 2100

    ladder RIN: 9.5

    RIN: 5.6 RIN: 2.8

    S. Fleige, V. Walf, S. Huch, MW. Pfaffl, 2005

  • RIN in different tissues and cell lines

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    liver (n = 22)heart (n =17)spleen (n =17)lung (n =22)rumen (n =23)reticulum (n =26)omasum (n =17)abomasum (n =17)ileum (n =17)jejunum (n =20)colon (n =19)caecum (n =16)lymph node (n =26)kidney cell (n = 3)corpus luteum (n = 5)granulosa cell (n = 5)oviduct (n = 5)WBC (n =5)

    RIN

  • Degradation scale

    18 S 28 S

    Marker WBC RIN: 9.6

    total RNA extracted from bovine tissues

    total RNA extracted from bovine tissues

    analyzed in Bioanalyzer analyzed in Bioanalyzer

    degradation scale ofmixtures between

    good and bad samples

    degradation scale ofmixtures between

    good and bad samples

    analyzed in Bioanalyzer analyzed in Bioanalyzer

    Degradation

    28 S18 S

    Marker WBCRIN: 2.8

    S. Fleige, V. Walf, S. Huch, MW. Pfaffl, 2005

  • Degradation of tissue extracted total RNA

    RIN 10.0 9.2 9.2 8.6 8.1 7.8 7.2 6.7 6.0 5.0 4.4 4.0

    18 S

    28 S

    5 S

    The intensity of bands decreases with increasing degradation

  • bovine ileum total RNA

    28S18S

    -ActinIL-1

  • bovine ileum: CP = TOP

    y = -0,2036x + 8,6557R2 = 0,7612

    y = -0,1461x + 13,565R2 = 0,4903

    R2 = 0,8119

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    RNA Integrity Number

    CP

    = Ta

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    18S beta-actin IL-1 Linear (18S) Linear (beta-actin) Linear (IL-1)

    18S

    beta actin

    IL-1y = -0,2583x + 21,787

    gene level of significanceInterleukin-1beta P < 0.001

    beta-Actin P < 0.0118S P < 0.001

  • Relative Quantification in real time qRT-PCR

    without real-time PCR efficiency correction

    2 (- CP) REST, qBASE,qGene, LC software

    with real-time PCR efficiency correction

    relative quantification

    external calibration

    curve withoutany referenece

    gene

    normalisationvia one

    reference gene

    via reference gene index

    >3 HKG

    ROX

  • bovine ileum: delta CP (compared to beta-actin expression)

    y = -0.057x - 4.909R2 = 0.096

    n. s.

    y = -0.112x + 8.222R2 = 0.260P = 0.04

    -8,0

    -6,0

    -4,0

    -2,0

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    RNA Integrity Number

    delta

    CP

    18S IL-1 Linear (18S) Linear (IL-1) Linear (18S) Linear (IL-1)

    18S

    IL-1

  • Influence of total RNA quality on qRT-PCR CP (Ct)IL-1: Crossing Point

    14,0

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    0 1 2 3 4 5 6 7 8 9 10RNA Integrity Number

    Cro

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    oint

    Reticulum (E) Lymph nodes (E) Lymph nodes (P) Colon (P) Lung (E)Corpus luteum (P) Caecum (P) Spleen (P) Abomasum (P)

  • Influence of total RNA quality on qRT-PCR efficiency

    28S: Amplification

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    2,0

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    PCR

    Eff

    icie

    ncy

    Reticulum (E) Lymph nodes (E) Lymph nodes (P) Colon (P) Lung (E)Corpus luteum (P) Caecum (P) Spleen (P) Abomasum (P)

  • Influence of qRT-PCR product length on RINbeta-actin procuts in various lenghts

    1012141618202224262830

    1,5 2,5 3,5 4,5 5,5 6,5 7,5 8,5RNA Integrity Number

    Cro

    ssin

    g P

    oint

    66 bp 99 bp201 bp480 bp795 bp975 bp

    Threshold RIN = 5.0

  • PCR efficiency in dependence of RIN

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    RNA Integrity Number

    PC

    R E

    ffici

    ency

    66 bp

    99 bp

    201 bp

    380 bp

    795 bp

    976 bp

  • Comparison of Experion & Bioanalyzer 2100

  • Comparison of 18S/28S rRNA ratioExperion & Bioanalyzer 2100

    Experion

    y = 0.107x + 0.475R2 = 0.32

    Experion

    y = 0.085x + 0.005R2 = 0.43

    Bioanalyzer 2100

    y = 0.177x + 0.2346R2 = 0.47

    Bioanalyzer 2100

    y = 0.1201x + 0.092R2 = = 0.53

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    RNA Integrity Number RNA Integrity Number

    200 ng total RNA analysed 50 ng

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