Real-time qPCR Experimental Design Considerations

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Real-time qPCR Experimental Design Considerations

• DNA Analysis Facility
• User Educational Series
• December 11, 2009

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Real-time qPCRExperimental DesignTopics to be
covered

• Basic Experimental Design for real-time qPCR
  experiments
• Identify the sources of variation in these
  experiments
• Make recommendations

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Real-time qPCR Experimental DesignData to be
presented
Presented at qPCR Symposium 2009 San Francisco CA
Nov 9-10, 2009
Tichopad A, Kitchen R, Riedmaier I, Becker C,
Stahlberg A, Kubista M. Design and Optimization
of Reverse-Transcription Quantitative PCR
Experiments. Clinical Chemistry 200955
1816-1823
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Real-time qPCRExperimental DesignqPCR
Experiments

• Real-time qPCR has many applications
• Viral Load detection
• Genotyping
• SNP detection
• ChIP Assays
• miRNA analysis
• Gene expression studies
• Gene Expression experiments are typically
  designed to test a hypothesis that a difference
  in gene expression exists between groups of
  biological subjects exposed to different
  treatments.

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Real-time qPCRExperimental DesignqPCR
Experiments pre-qPCR steps

• Sampling
• Collection of samples
• Storage of samples prior to extraction
• Nucleic Acid Extraction
• Method of extraction
• Presence of inhibitors
• Storage of RNA prior to RT Reaction
• Nucleic Acid Quality and Quantification
• Check RNA Quality
• Good Quantification in order to balance RT Rxn.
• Reverse Transcription
• Selection of enzyme and priming strategy
• gDNA contamination?
• Presence of inhibitors?

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Real-time qPCRExperimental DesignqPCR Step

• Real-time qPCR
• Assay validation
• Choice of Chemistry
• Choice of primers/probes
• PCR efficiency
• Dynamic Range of Assay
• Choice of Endogenous Control
• All of these steps impact the end result of the
  qPCR measurement, and they all have the potential
  to add noise to the experimental data.

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Real-time qPCRExperimental DesignSources of
Variation

• Studied Variance
• The treatment effect can only be resolved if it
  is larger than the random noise within the groups
  due to the confounding noise.
• Confounding Variance
• Biological or Inter-subject Variance
• This is the random difference between individuals
• Processing Variance
• These are technical variances due to processing
  of samples, extractions, RT and qPCR reactions.

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Real-time qPCRExperimental DesignGoals of
Experimental Design

• Goal of Experimental Design is to optimize your
  treatment effect relative to the confounding
  effect of your biological and processing noise.
• This requires knowing where your sources of
  variation are likely to occur and accounting for
  these with you data analysis.
• Being cost effective with your choices.

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Real-time qPCRExperimental DesignDetermine
Sources of Variation
Kubistas group designed an experiment to look at
the sources of variation that are found in a
typical qPCR experiment.
Tichopad et al. Design and Optimization of
Reverse-Transcription Quantitative PCR
Experiments. Clinical Chemistry 200955
1816-1823
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Real-time qPCRExperimental DesignDetermine
Sources of Variation

• Liver Tissue
• qPCR Assays ACTB, IL1B, CASP3, FGF7
• Blood
• qPCR Assays ACTB, IL1B, CASP3, IFNG
• Cell Cultures
• qPCR Assays ACTB, H3F3A, BCL2, IL8
• Single Cells individual astrocytes from mouse
  brain
• qPCR Assays 18s

Tichopad et al. Design and Optimization of
Reverse-Transcription Quantitative PCR
Experiments. Clinical Chemistry 200955
1816-1823
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Real-time qPCRExperimental DesignStatistical
Analysis
Total Variance Variance contribution from
processing steps Subject Sample/Extraction RT qP
CR
Tichopad et al. Design and Optimization of
Reverse-Transcription Quantitative PCR
Experiments. Clinical Chemistry 200955
1816-1823
12
Real-time qPCRExperimental DesignDetermine
Sources of Variation
Total noise SD Cumulative variance which is
expressed as the SD of measured CT values.
Highlighted figure is the mean of all 4 genes.
Tichopad et al. Design and Optimization of
Reverse-Transcription Quantitative PCR
Experiments. Clinical Chemistry 200955
1816-1823
13
Real-time qPCRExperimental Design Sources of
Variation Liver samples

• 3 subjects x 3 samples x 3 RTs x 3 qPCRs (81
  CTs measured)

Subject Level SD was negligible at this
step Sampling Level Largest SD was estimated
for this step. Mean SD1.2 Ct which is gt2 fold
variation. RT Level 3 genes mean SD0.39CTs
4th gene SD 0.9 CTs qPCR Level showed
highest reproducibility. Mean SD0.09 CTs
Total noise SD estimate 1.5 CTs
Tichopad et al. Design and Optimization of
Reverse-Transcription Quantitative PCR
Experiments. Clinical Chemistry 200955
1816-1823
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Real-time qPCRExperimental Design Sources of
Variation Single cells

• 3 subjects x 3 samples x 1 extractions x 3 RTs x
  3 qPCRs
• (81 CTs measured)

Subject Level SD was negligible. Sampling
Level SD1.9 CTs This is consistent with
other studies that show mRNA levels vary greatly
between individual cells. RT Level SD0.30
CTs qPCR Level SD0.51 CTs
Total noise SD estimate 2.0 CTs
Tichopad et al. Design and Optimization of
Reverse-Transcription Quantitative PCR
Experiments. Clinical Chemistry 200955
1816-1823
15
Real-time qPCRExperimental Design Sources of
Variation Blood samples

• 3 subjects x 1 samples x 3 extractions x 3 RTs x
  3 qPCRs
• (81 CTs measured)

Subject Level Negligible for 2 genes, SD1CT
for other 2 genes. Sampling Level Highest
reproducibility SD0.12 CTs RT Level Similar
for all genes SD0.24 qPCR Level 3 higher
expressors (CTs 16-25) SD0.17 CTs Low
expressor SD0.4 CTs
Total noise SD estimate 0.66 CTs
Tichopad et al. Design and Optimization of
Reverse-Transcription Quantitative PCR
Experiments. Clinical Chemistry 200955
1816-1823
16
Real-time qPCRExperimental Design Sources of
Variation Cell Cultures

• 1 subject x 10 samples x 1 extraction x 3 RTs x
  3 qPCRs
• (90 CTs measured)

Subject Level Cell cultures are unique at this
level due to their clonal nature. Sampling
Level mean SD0.27 CTs RT Level mean SD0.31
CTs qPCR Level mean SD0.14 CTs
Total noise SD estimate 0.44 CTs
Tichopad et al. Design and Optimization of
Reverse-Transcription Quantitative PCR
Experiments. Clinical Chemistry 200955
1816-1823
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Real-time qPCRExperimental Design qPCR Variance

• qPCR variance (mean SD0.13 CTs) is lower than
  the variance of other steps and does not depend
  on sample type.
• qPCR variance will be higher in samples with CTs
  gt 30.
• qPCR was done in duplicate in most publications,
  but without justification as to why selected.
• The use of single wells is indicated but does not
  insure against a failed reaction.
• If cDNA is limited, a single qPCR well is
  preferable because splitting into two wells will
  further reduce the cDNA available in the qPCR
  reaction.

Tichopad et al. Design and Optimization of
Reverse-Transcription Quantitative PCR
Experiments. Clinical Chemistry 200955
1816-1823
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Real-time qPCRExperimental Design Kubista
General Recommendations
Supplemental Table 1
General Recommendation Upstream replicates are better than downstream replicates. Hence generally, including more subjects is superior to any other replicates and should be preferred as long as it is economically feasible.
Solid tissue Several samples should be withdrawn from the same tissue and processed separately (sampling replicates). Other types or replicates are inferior
Blood Producing RT replicates is superior to any other types of replicates.
Cell culture The number of cell culture wells should be maximized prior to any other type of replicates. Secondarily, increasing the number of RT replicates should be considered.
Low copy transcript Replicates should be produced at the RT level rather than at any other.
Tichopad A, Kitchen R, Riedmaier I, Becker C,
Stahlberg A, Kubista M. Design and Optimization
of Reverse-Transcription Quantitative PCR
Experiments. Clinical Chemistry 200955
1816-1823
19
Real-time qPCRExperimental Design Conclusions

• Perform a fully nested pilot study to identify
  the sources of variation associated with your
  experiment.
• Cost-optimize the experimental design to include
  the optimal number of subjects and technical
  replicates you need to strengthen the power of
  your experiment.

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Real-time qPCRExperimental Design Questions?