1. Principles and Important Considerations
Real Time PCR
Principles and Important Considerations

2. Chemistry

3. Chemistry

4. Chemistry

6. 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).

7. 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

8. 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.

9. Always use during analysis
Amplification curve:
log view
Always use during analysis
Plateau
Exponential
Linear
Amplification curve:
linear view

10. It's all about Ct Ct
Threshold

11. Threshold set too high
Threshold set too low

12. 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

13. 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)

14. Bad Comparative Ct example1
Bad Comparative Ct example 1 2 3
Too much variation in endogenous control Ct values
Sigmoidal curves. Check baseline levels
No amplification

15. 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≥ /230 ≥2.0)
At least 3 biological replicates

16. 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!

17. Reaction setup ALWAYS set your plates/tubes on a rack
NEVER set your plates/tubes directly on the bench or ice

18. Keep the machine clean and free of dust!
Plates go directly on the heating block.
Tubes go on a black rack.

19. Online help
Real-Time PCR Systems. Chemistry Guide: ments/cms_ pdf
Guide to Performing Relative Quantitation of Gene Expression Using Real-Time Quantitative PCR:
nts/cms_ pdf
Troubleshooting guide: trouble-shooting-guide.html