Real Time PCR • Principles and Important Considerations
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
Referencesample • 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 Always use during analysis Plateau Exponential Linear Amplification curve: linear view
Ct It's all about Ct Threshold
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
Endogenous control Gene of interest Good Comparative Ct example (for gene expression) • Small variation in endogenous control Ct values • Shape of curve, including linear phase (log view)
1 1 2 2 3 Bad Comparative Ct example • Too much variation in endogenous control Ct values • Sigmoidal curves. Check baseline levels • No amplification
Other important considerations • Really sensitive technique: 1 DNA molecule can be detected • Contaminationand 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 NEVER set your plates/tubes directly on the bench or ice ALWAYS set your plates/tubes on a rack
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/generaldocuments/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/generaldocuments/cms_042380.pdf • Troubleshooting guide:http://www.appliedbiosystems.com/absite/us/en/home/support/tutorials/realtime-pcr-trouble-shooting-guide.html