Download PCR settings, pitfalls and artefacts

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PCR settings, pitfalls and artefacts
Dr. Sci. Sabine Franke
May 17th 2013
Molecular Biology and Cytometry Course
SCK CEN - Mol
Overview
•  Qualitative PCR
Reagents
Primers
Thermocyclers
Controls
Nested PCR
•  Quantitative PCR
Qualitative PCR
PCR optimization
•  sufficient and specific amplification.
Problem
•  Low yield or no amplification product
•  Multiple, nonspecific amplification
products
Check basic parameters
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template quality
template quantity
PCR primer design
reverse transcription
magnesium concentration
cycling parameters
reaction buffer composition
enzyme concentration
cycling parameters
PCR additives
Thermocyclers
others
Template quality
•  RNA quality and purity is crucial for
success
•  Extraction and storage of RNA important
Template quantity
•  Avoid differences in RNA quantity
PCR primer design
•  Length ranging from 15–30 bases
•  G+C content 40–60%
•  Avoid internal secondary structure (primer dimers/
hairpins)
•  Avoid three G or C nucleotides in a row near the 3´-end
•  Ideally, both primers should anneal at the same
temperature. Annealing temperature
starting ~ 5°C below calculated Tm
•  Purity of primers (desalted, HPLC,…)
PCR primer design
Nonspecific priming - Secondary products
and lack of specificity
Primer dimer formation – Lower yields
Lower sensitivity
IDT,Eurogentec, Promega,….
BLAST search; eventually other primer
Reverse transcription
Different reverse transcriptases have
significantly different efficiencies.
Reverse transcription
One step RT
One step RT PCR is
performed sequentially in
the same tube using the
entire amount of cDNA synthesis
products as the PCR template.
Two step RT
Two step RT-PCR are performed sequentially,
but only a portion of the cDNA products is
used as the template for PCR, which is performed
in a separate tube.
Reverse transcription
•  One or two step RT-PCR
One-step RT-PCR more sensitive and
requires less pipetting
Two-step RT-PCR allows multiple PCRs
from a single RT reaction to quantify
multiple targets or perform replicate
assays.
Reverse transcription
•  amount of RNA
•  RNA quality and purity can affect
first-strand cDNA synthesis efficiency
RNA degradation -> efficientcy RT reaction
Magnesium concentration
•  Effects of magnesium concentration on
PCR amplification
Magnesium is important cofactor to help
stabilization of DNA structure.
Cycling parameters
Number of cycles: 25-40 cycles
Reaction buffer composition
Choise of nucleotides
Enzyme concentration
•  Concentration influences
PCR reaction
PCR additives
Has to be checked for each protocol
(example DMSO)
Thermocyclers
•  Calibration - thermocyclers can loose
calibration over time
•  Some blocks on thermocyclers do not
support “fast cycling”
•  Older thermocyclers need mineral oil layer
over reactions
Others
•  DNA contaminants (labo area)
•  plastics
Problem:
No Product
Thermocycler
Issue
X
[MgCl2]
XX
Template/
Primer
X
Cycling
Conditions
X
Enzyme
X
Reagents
XX
Cloning Issues
XX
XX
Wrong Sequence
Non-Specific
XX
XX
X
XX
XX
Troubleshooting
Weak or no amplification:
increase # cycles
increase time at step(s)
increase quantity of template
increase Taq concentration by 2X
try different Taq (e.g. Faststart)
increase MgCl2, primers, dNTPs
Smears above band
decrease # cycles
decrease concentration of reagent(s)
Sensitivity problem
Certified reference material
cell line documentation and
verification
Troubleshooting
low weight bands:
decrease MgCl2 concentration
decrease # cycles
raise annealing temperature
decrease primer concentration
decrease dNTPs
high weight bands
decrease Taq concentration
Prevent
How?
Bad sequence
Perform a bioinformatic evaluation
Contamination
separate RNA area
sample prep, mix set-up, PCR
flow
Contamination
Add DNA or RNA to your wells last
cross contamination
filter tips (aerosol-resistant)
freeze/thaw cycles
Make aliquots
Bubbles
Centrifuge PCR plate before loading
Controls
Control requests
• Plasmids work as control templates but can
have slightly different conditions compared to
gDNA or cDNA
•  If possible:
Certified reference material
(cell line documentation)
•  negative control(s): check for contamination
•  Run a positive control (a sample known to
amplify well)
Control
•  Include an internal standard in your
PCRs
•  second primer pair that amplifies a
“housekeeping” gene can be
included in the reaction
•  Participating in external QC rounds
Nested PCR
Nested PCR
Attention
•  Special care for nested PCR!
More sensitive, but …
•  opening increases the risk of contaminating
subsequent reactions with amplified product.
•  Never opening more than one tube at a time
•  Using a separate thermocycler
•  Adding additional negative control
Quantitative PCR
De gekoppelde afbeelding kan niet worden weergegeven. Het bestand is mogelijk verplaatst, heeft een andere naam gekregen of is verwijderd. Controleer of de koppeling verwijst naar het juiste bestand en de juiste locatie.
Quantitatif PCR
design
Quantif.
samples
analysis
assays
guidelines
Ref.gene
Quantitative PCR
•  Include an internal standard in RT-PCRs.
“housekeeping” gene
•  Housekeeping genes can help account for
differences in the amounts of starting
nucleic acid.
Tips
Change positions of controls
Tips
Pipette calibration frequently
Tips
•  Do RNA extraction in one serie
•  Do RT step in one serie
Tips
Participating in QC rounds not
only for tests but also for
extraction/storage conditions
Housekeeping gene
•  Include an internal standard in RT-PCRs.
For example, a second
primer pair that amplifies a “housekeeping”
gene can be included in the reaction.
(constant expression levels among
the samples compared)
Housekeeping gene
•  Stable expression
•  Should be expressed in all cells
•  HK gene should have the same copy
number in all cells
•  Transcription level in same range as target gene
Perfect standard does not exist
Gene expression
Gene expression result: impact of RNA quality
Accuracy of gene expression is highly
dependent onmRNA quality
Reference gene expression stability is
influenced by RNA quality
Quantitative RT-PCR partially degraded RNA may not
give accurate representation of gene expression
Standard-curve method
Absolute and relative quantification
Interpretation/quantification outside
standard curve
Efficienty
•  Control the efficientcy of the reaction
Variables which can affect the efficiency:
- Length of the amplicon
- Secondary structure
- Primer design
Conclusion
•  check working area before starting
•  check primers before starting
•  check protocol before starting
•  check settings (cycles, baseline, threshold)
Before starting
•  use controls
•  analyse problems step for step
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