Principles and Important Considerations 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.

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

It's all about Ct 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

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)

Bad Comparative Ct example 1 Bad Comparative Ct example 1 2 3 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 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-pcr- trouble-shooting-guide.html