Real time RTPCR

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Real time RT-PCR

• Joshua Gray
• Pharmacology Toxicology
• Rutgers University
• gray_at_rci.rutgers.edu
• www.rci.rutgers.edu/gray

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Basics of real time RT-PCRAbundance of mRNA

• Central hypothesis Changes in gene expression
  reflect changes in cell function
• Nearly all physiological changes in animals are
  accompanied by changes in gene expression
• Immune response
• Toxic response
• Pharmacological response

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Outline

• Basics of PCR and real time PCR
• Experimental Design
• Data Analysis
• Application to Drug Development and Design

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Polymerase Chain Reaction

• In vitro method for the amplification of short
  (up to 5000bp) pieces of DNA
• Relies on a thermostable form of DNA polymerase
• Thermus aquaticus

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Polymerase Chain Reaction
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Polymerase Chain Reaction

• Required reagents
• Template DNA
• Primers
• DNA polymerase
• dNTPs
• Thermocycler

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Polymerase Chain Reaction

• Electrophoresis

Ethidium bromide
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Polymerase Chain Reaction

• Proper controls
• Water blank
• Concentration curve
• Internal standard control
• Genomic DNA contaminant control

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Internal standard
Gene of interest
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Polymerase Chain Reaction

• Advantages
• Sensitive
• Versatile easy to test new genes
• Primers are inexpensive
• Reliable
• Much more than microarrays for individual
  transcripts
• Standardized competitor templates or standard
  curves
• Allow comparison between expts
• Internal standards
• Addresses variation in tissue starting amounts or
  loading errors

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Polymerase Chain Reaction

• Disadvantages
• Optimizations required
• Annealing temperature
• Number of cycles
• Small order of magnitiude sensitivity for
  detection

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Real time PCR

• Similarities to regular PCR
• Same use of primers, template, enzyme, and
  nucleotides
• Differences from regular PCR
• Special machine
• Optical dyes
• Optimizations

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Real time PCR

• Abbreviation problems
• RT-PCR reverse transcription polymerase chain
  reaction
• Do not label real time PCR as RT-PCR
• Real time RT-PCR is RT-PCR, using a real time PCR
  system

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Real time PCR

• Real time PCR machine 7900 HT

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Real time PCR

• Fluorescent detectors
• SYBR green Applied Biosystems
• TaqMAN Applied Biosystems
• LUX Invitrogen
• Annealing phase method - FRET method,
  non-quenching
• New Exiqon Probe Library

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Real time PCR

• SYBR green
• Binds to double-stranded DNA
• Fluorescence increases 20X upon binding
  double-stranded DNA
• Cannot discriminate between desired versus
  undesired products
• Cheapest method for PCR
• Look for increases in fluorescence with increased
  product

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Real time PCR

• TaqMan
• Gold standard for real-time PCR
• Uses three primers
• Normal two primers for amplification
• Third primer binds internal to the product
• The third primer
• Contains two fluorescent compounds a fluor and
  a quencher
• Third primer is degraded by the exonuclease
  activity of Taq
• Reduces the FRET (fluorescence resonance energy
  transfer) of signal from the fluor to the quencher

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Real time PCR

• LUX light upon extension
• probe is quenched when in hairpin form
• Extended form has brighter signal
• Incorporated probe as the brightest signal
• Look for increased fluorescence with increased
  product

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Real time PCR

• Annealing phase method
• Also uses three probes
• One of the two regular probes is labeled with a
  fluor
• Third probe also has a fluor
• Annealing of first and third probes allows FRET,
  during the annealing stage
• Look for increase in fluorescence

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Real time PCR

• Quenching Method
• Similar to TaqMan, but instead of a quencher, we
  look for a decrease in fluorescence of the second
  fluor

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Real time PCR

• Exiqons Probe Library
• Similar to TaqMan, but the internal dual-labeled
  probes are 8-mers
• 90 different octamers in the 3500 kit
• Enough for 900 reactions each
• Can reorder more for 180 of individuals, or buy
  separately
• Andrew Brooks
• Online primer design

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Real time PCR

• Uses of real time PCR
• Gene expression analysis
• Splice variant detection
• Viral load
• Mutation analysis

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RNA preparation

• Methods
• Trireagent (Trizol, others), cheap, low quality
  requires clean-up
• RNeasy - 3 per reaction

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Outline

• Basics of PCR and real time PCR
• Experimental Design
• Data Analysis
• Application to Drug Development and Design

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Experimental Design

• Replicates of your samples
• Standard curve
• Water blank
• RNA/genomic DNA control
• Dissociation curve

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Experimental Design

• Replicates of your samples
• Real replicates versus artificial
• True replicates are from different samples
• It can be helpful to run your sample more than
  once, to check for pipetting error
• Good for small differences in gene expression
• Expensive

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Pipette Error
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Pipette Error

• Pipet as large a volume as you can to avoid pipet
  error
• Dilute your samples, when possible, to be able to
  pipet larger volumes

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Experimental Design

• Standard curve
• Pooled group of a random set of samples
• Serially diluted 12, five times
• Determines the efficiency of the reaction
• PCRs ideally are 100 efficient, if no reagents
  are limiting
• TaqMan claims 100 efficiency, and no need for a
  standard cruve
• Low copy numbers are not necessarily 100
  efficient

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Experimental Design

• Water blank
• To determine if you have primer dimers in your
  reaction
• SYBR green and other two primer-based methods can
  have primer dimers
• TaqMan, other probe-based amplifications are
  immune to this

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Experimental Design

• RNA/genomic DNA control
• RNA purification often includes some genomic DNA
  contaminant
• Genomic DNA can influence your results if
• Your primers do not overlap a intronexon site
• Genomic DNA contamination makes your
  non-expressing controls look higher than they
  should be
• Removing genomic DNA will improve your results

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Experimental Design

• Dissociation curve (SYBR green only)
• Slowly heats the samples from 60C
  (annealing/elongation temperature) to 95C
• Looks for changes in fluorescence at each
  temperature
• Since different PCR products tend to dissociate
  at different temperatures, this method will tell
  you whether you have multiple products in your
  sample

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Experimental Design

• Considerations
• Problems with real time PCR are usually due to
  problems with nucleic acid contamination, RNA
  quantity, reverse transcription
• Start with equal amounts of RNA for the Reverse
  transcription step
• Pipetting error can dramatically contribute

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Outline

• Basics of PCR and real time PCR
• Experimental Design
• Data Analysis
• Application to Drug Development and Design

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Data analysis

• Things to check after your first run with a new
  primer set
• Standard curve
• Water blank
• Dissociation curve
• RNA sample (to check genomic DNA contamination)

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Data analysis

• NCT Number of DNA molecules
• at Cycle of Threshold (CT)
• N0 Initial number of DNA molecules
• E PCR Efficiency (0ltElt1)
• NCTN0(1E)CT

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Standard Curve
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Not-so-good Standard Curve
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Lipocalin low RNA concentration example
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Dissociation Curve
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Dissociation curveFirst Derivative
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Rule of Seven

• If there is primer dimer formation, or genomic
  DNA contamination, you may still be OK
• The contamination must be at least 7 cycles away
  from your samples
• Assuming 100 efficiency, this contaminant would
  then be lt5 of your total product

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Data analysis
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Data analysis

• Next, assign values to your standard curve
• This curve is designed to give us efficiency for
  amplification
• Samples do not have to fall within this standard
  curve to be analyzed
• The first tube has twice as much starting
  material as the second.
• Tube 1 1000 (unitless number)
• Tube 2 500
• Tube 3 250
• Tube 4 125
• Tube 5 63.5

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Data analysis

• Tube 6, the water blank, never crosses the
  threshold. Therefore, you cannot give it a value
  or include it in the curve

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Data analysis

• Choosing appropriate controls
• Common controls are GAPDH, beta-actin, and 18s
  rRNA. Housekeeping genes
• Good controls are not regulated by your
  treatment(s)
• Each control has its own pitfall
• rRNA different RNA polymerase, expressed very
  high relative to samples
• GAPDH and actin may be regulated by some
  treatments
• I usually run two controls and check them against
  each other

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Comparison of actin and GAPDH in Mouse Tissue
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Data analysis

• Correct your primers results against the control
• (IL-6)/(actin)
• Look for fold induction versus the non-treated
  control (set that number to 1)
• You do not get RNA quantity only changes in
  fold amount

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Data Example
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Data example
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Data analysis

• A word about delta-delta Ct method
• This was the first method to quantify real time
  PCR products
• Includes assumptions, like 100 efficiency of
  reaction
• 2(? ? Ct)

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Outline

• Basics of PCR and real time PCR
• Experimental Design
• Data Analysis
• Application to Drug Development and Design

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Case Study

• Paraquat poisoning of mice
• Ingested paraquat is a tissue-specific toxicant
• Lung fibrosis, death
• Are there other target tissues?
• What are the genes involved in the regulation of
  paraquat poisoning?

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Case study

• Inflammatory
• ICAM
• INOS
• TGFbeta
• IFNgamma
• TNFalpha
• IL-10
• IL-6
• IL1beta

• Prostaglandins/Leukotrienes
• 5-lipoxygenase
• 12-lipoxygenase
• LTA4
• Other markers
• PPARgamma
• STAT-1
• Myeloperoxidase
• NADPH oxidase

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Case study

• Compare the controls
• Actin and GAPDH

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(No Transcript)
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Correlation between control genes
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Conclusions

• See my webpage for a complete tutorial on real
  time PCR
• www.rci.rutgers.edu/gray
• gray_at_eohsi.rutgers.edu
• Pitfalls of QRTRT-PCR. Bustin et. al.
• http//jbt.abrf.org/cgi/content/full/15/3/155
• Accurate normalization of real-time quantitative
  RT-PCR data by geometric averaging of multiple
  internal control genes
• http//www.pubmedcentral.nih.gov/articlerender.fcg
  i?toolpubmedpubmedid12184808
• RNA preparation
• http//www.abrf.org/Other/ABRFMeetings/ABRF2002/RN
  Aprimer.pdf