Real Time PCR
Principles and Important Considerations
Chemistry
Chemistry
Chemistry
Primer/Probe design is really important
• Use PrimerExpress
• No mismatches are allowed (make sure you have the
correct sequence)
• MGB labeled with VIC or FAM (never ROX)
• Primers/Probe need to be validated
• For gene expression experiments it is ideal that one of
the primers or probe sits in an exon junction (prevents
amplification from genomic DNA).
Endogenous control gene:
• Present in all experimental samples
• Expression does not vary between treatments,
tissues, age, etc. i.e. constant expression levels
• By using an endogenous control as an active
reference you can normalize quantification of
mRNA target for differences in the amount of
total RNA added to each sample. i.e. loading
control
• Commonly used: 18S or 25S rRNA, actin,
GAPDH, ubiquitin, etc
Reference sample
•
Used in Comparative CT and relative standard curve
experiments
•
Sample used as the basis for relative quantitation
results. i.e. Everything gets expressed and compared
relative to this sample.
•
Also called calibrator
•
It doesn’t matter which sample is used, however
normally the negative control is used.
Amplification curve:
log view
Linear
Exponential
Always use during analysis
Plateau
Amplification curve:
linear view
It's all about Ct
Threshold
Ct
Threshold set too high
Threshold set too low
Threshold is important
• Threshold determines Ct values
• Ct values are used to calculate relative
expression, presence/absence, etc. It's all
about Ct values
• Threshold should be set in the linear portion
(parallel lines) in log view. Always check!
• Do not use Ct values of 35 or higher. Repeat
using more cDNA/DNA
Good Comparative Ct example
(for gene expression)
Endogenous control
Gene of interest
• Small variation in endogenous control Ct values
• Shape of curve, including linear phase (log view)
1
Bad Comparative Ct example
1
2
2
3
1) Too much variation in endogenous control Ct values
2) Sigmoidal curves. Check baseline levels
3) No amplification
Other important considerations
• Really sensitive technique: 1 DNA molecule can be
detected
• Contamination and cross-contamination IS a problem
• Always include no template (water) controls for each gene in
each plate
• Include no RT control (RNA) some of the time. Specially when
you are starting using this technique
• RNA/cDNA/DNA quality is critical. OD values (260/280≥ 1.8
260/230 ≥2.0)
• At least 3 biological replicates
Good Practices
• Mortars and pestles should be treated with bleach and
autoclaved
• Pipettes and centrifuge should be decontaminated (RNAase
Away)
• Cover your working area and replace mats after each round of
extractions
• Change gloves frequently
• If extracting RNA don't talk to your samples! RNases are present
in your saliva. Use RNase-free water
• Keep your working area clean!
Reaction setup
ALWAYS set your
plates/tubes on a rack
NEVER set your plates/tubes
directly on the bench or ice
Keep the machine clean and
free of dust!
Plates go directly
on the heating
block.
Tubes go on a
black rack.
Online help
• Real-Time PCR Systems. Chemistry Guide:
http://www3.appliedbiosystems.com/cms/groups/mcb_marketing/documents/generaldocu
ments/cms_041440.pdf
• Guide to Performing Relative Quantitation of Gene Expression Using Real-Time
Quantitative PCR:
http://www3.appliedbiosystems.com/cms/groups/mcb_support/documents/generaldocume
nts/cms_042380.pdf
• Troubleshooting guide:
http://www.appliedbiosystems.com/absite/us/en/home/support/tutorials/realtime-pcrtrouble-shooting-guide.html