Analysis of data from Real time experiments

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Analysis of data from Real time experiments
J.M.K. Mulema Department of Molecular and Cell
Biology University of Cape Town
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Introduction

• Quantifying RNA Northern blotting, In situ
  hybridization, RNAse protection assays,
  Microarray, RT-PCR.
• Real time PCR
• Data collected throughout the PCR process as it
  occurs.
• Amplification and detection combined into a
  single step.
• Reactions characterized by the point in time
  where the target amplification is first detected.
• Cycle threshold (Ct), the time at which
  fluorescent intensity is greater than background
  fluorescence.
• Greater starting target DNA, faster significant
  increase in fluorescent, lower Ct
• Requires much less RNA template

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One step Vs Two step

• Two step
• Reverse transcription and PCR occur in separate
  tubes
• Allows several different real time PCR assays on
  dilutions of a single cDNA.
• Reactions from subsequent assays have the same
  amount of template to those assayed earlier.
• Date from two step is quite reproducible.
• Allow for increased DNA contamination. Design the
  target PCR product to span introns

• One step
• cDNA synthesis to PCR amplification is performed
  in a single tube.
• Minimizes experimental variation because both
  enzymatic reactions occur in a single tube
• Uses RNA starting template. Prone to rapid
  degradation if not handled well.
• Not suitable in situations where the same sample
  is assayed on several occasions over a period of
  time.
• Less sensitive than two step protocols.

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Identify genes that play a role in resistance
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• At3g51660 (Macrophage migratory)
• At4g30270 (MERI-5 protein)
• At2g47190 (Myb family transcription)
• At5g39610 (No Apical Meristem)
• At5g06860 (PGIP1)
• At4g24340 (Phosphorylase)
• At5g07010 (Sulfotransferase)
• At1g22400 (UDP-glucoronosyl)
• At1g62300 (WRKY)
• At3g04720 (Hevein-like protein)
• At3g28930 (avrRpt2-induced protein)
• At3g50480 (Broad spectrum)
• At2g24180 (CYP 450)
• At3g04220 (Disease resistance protein)
• At1g52200 (Expressed protein)
• At2g39030 (GCN5-related acetyltransferase)
• At4g16260 (Glycosyl hydrolase)
• At4g15610 (Integral membrane protein)
• At4g33150 (lysine-ketoglutarate)

• Arabidopsis leaves infected with B. cinerea
• Extracted RNA from first three replicates
  (untreated, 12 hrs pi and 24 hrs pi)
• Made overnight cDNA synthesis
• Used Superscript III reverse transcriptase in a
  20µl reaction.
• Diluted the synthesized cDNA 1 in 10
• Designed primers to amplify products ranging from
  70-150 bp
• Amplification optimized with a conventional PCR
  before real time.

• At4g10340 (Chlorophyll A-B)
• At1g72610 (Germin-like protein)
• At1g12900 (GAPHD)
• At5g38430 (RUBSCO)
• At1g20340 (Plastocyanin)

• At5g25760 (Ubiquitin-conjugating enzyme)
• At5g06600 (Ubiquitin-specific protease)
• At1g04820 (Tubulin alpha 2-alpha 4)
• At5g44200 (Nuclear cap-binding protein)

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• Absolute/Relative quantification
• Used serial diluted standards of known
  concentration to generate a standard curve.
• Standard curve is a linear relationship between
  the ct and the initial amount amounts of RNA or
  cDNA.
• This allows the determination of the
  concentration of unknowns based on their ct
  values
• Assumes that all standards and samples have equal
  amplification efficiencies.
• The concentrations of serial dilutions should
  encompass all samples and stay within the range
  that can be quantified

Generating a standard curve
Pooled sample
Dilution 1 (10-1) Dilution 2 (10-2) Dilution 3
(10-3)
Carry out runs in triplicates
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Upregulated genes
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Down regulated genes
Reference genes (Relative Conc)
Reference genes (Ct values)
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Types of real-time quantification

• Absolute quantification
• Uses serially diluted standards of known
  concentration to generate a standard curve.
• PCR standards
• Fragment of double stranded DNA
• Single stranded DNA
• Complementary RNA bearing the target sequence
• Relative quantification
• Changes in gene expression are an external
  standard or calibrator
• Two standard curve method
• Comparative Ct (Delta Delta Ct) method
• Pfaffl method Relative expression software tool
  (REST)

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Calculation of relative values (At2g24180)
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Relative expression software tool (REST)

• Purpose is to determine if there is a significant
  difference between samples and controls taking
  into account issues of reaction efficiency and
  reference gene normalization.
• Randomization are used in Rest.
• The alternate hypothesis P(H1) is based on the
  assumption that the difference between sample and
  control groups is due only to chance.
• REST performs 50,000 random reallocations of
  samples and controls between the groups and
  counts the number of times the relative
  expression of the randomly assigned group is
  greater than the sample data
• Concentration efficiencyavg(Controls)
  avg(Samples)
• Expression goiConcentration refConcentration
• Expression GEOMEAN(goiConcentration refConc1,
  goiConcentration refConc2, )
• Calculate a normalization factor equal to the
  geometric mean
• REST performs its calculations based on CP and
  efficiency values determined by standard curve or
  kinetic techniques.

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Delta Delta Ct method

• Xn Xo x (1 Ex)n
• XT Xo x (1 Ex)CT,X KX
• RT Ro x (1 ER)CT,R KR
• Dividing XT by RT gives the expression
• XT Xo x (1 Ex)CT,X KX
• RT Ro x (1 ER)CT,R KR
• Assuming equal amplification efficiencies
• EX ER E
• Xo
• Ro

Calibration
Amount of the target normalized to an endogenous
reference and relative to the calibrator is given
by Amount of target 2-??CT
Assumption The amplification efficiency of the
target and reference are approximately equal
x (1 E) CT,X-CT,R
XN x (1 E) ?CT
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Conclusion

• Real Time PCR is a powerful technique that gives
  quantitative answers difficult to obtain with end
  point PCR, however, all steps need to be
  controlled from sampling to PCR including
  manipulations like extraction and reverse
  transcription