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Learn how to compare gene expression levels between two samples using qPCR analysis. Get step-by-step instructions and necessary protocols for both Absolute qPCR and Relative qPCR methods.
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Ref2 Sample 1 • Ref1 Sample 1 • Ref3 Sample 1 • Ref3 Sample 2 • Ref1 Sample 2 • Ref2 Sample 2 • GOI Sample 1 • GOI Sample 2 • Standard 6 • Standard 5 • Standard 4 • Standard 2 • Standard 1 • Standard 3 • Ref1 St 4 • Ref2 St 3 • Ref1 St 1 • Ref1 St 2 • Ref1 St 3 • Ref2 St 1 • Ref3 St 1 • Ref3 St 2 • Ref3 St 3 • Ref3 St 4 • Ref2 St 4 • Ref2 St 2 • GOI St 3 • GOI St 4 • GOI St 2 • Sample B • NTC • GOI St 1 • Experiment • Relative qPCR • Absolute qPCR • you need to provide: • 1. RNA or cDNA from two samples you want to compare (sample 1 and sample 2) • 2. sequences of primers for one gene of interest (GOI) and three reference genes • you will need to: • estimate efficiency of your primers • find out the amount of your GOI in sample 1 compared to sample 2 • you will be provided with: • 1. sample A: plasmid of known concentration • 2. forward and reverse primers to detect the plasmid • 3. %E of the primers • 4. sample B:the same plasmid with unknown concentration • you will need to: • find out the concentration of the sample B
Kits and Protocols For qPCR we will use DyNAmo Flash SYBR Green qPCR Kit (# F-415L, ThermoScientific.follow the link for Manual and other info) • For RT we will use Maxima First Strand cDNA Synthesis Kits for RT-qPCR (# K1642, ThermoScientific. follow the link for Manual and other info)
Absolute qPCR • Use sample A to make 6 serial 10 fold dilutions. • Take into consideration: • each dilution will be checked in 3 identical PCR reactions (technical replicates, a control for pipetting error) • you will use 5 ul of template for each PCR reaction • sample A: 50 ul of plasmid, concentration 10 ng/ul • 5 uM forward primer (F) • 5 uM reverse primer (R) • sample B: the same plasmid, concentration is unknown • NTC= no template control= water • 2. Calculate the amount of qPCR reactions required and prepare corresponding volume of the Master mix (amount of reactions + 2) • 3. Pipet the Master mix into the plate (15 ul /well) • 4. Add assigned templates to the Master mix in the wells • 5. Seal the plate with the film. store at 4oC before run • 6. Program the run • 7. Perform the run • 8. Your data file will be uploaded on the course web site • 9. Analyse the data • 10. Write a report • 11. Make a presentation
Relative qPCR • 1. Pool together x+y ul of our cDNA samples. If necessary dilute it up to 30 times (call this sample ”cDNA pool”). • Prepare a four serial dilutions of the cDNA pool • Take into consideration: • each dilution will be checked in 3 identical PCR reactions (technical replicates, a control for pipetting error) • you will use 5 ul of template for each PCR reaction • try not to dilute your initial cDNA samples more than 300 times • sample 1: cDNA of your choice • sample 2: another cDNA of your choice • 5 uM Ref1 primers (forward and reverse) • 5 uM Ref2 primers (forward and reverse) • 5 uM Ref3 primers (forward and reverse) • 5 uM GOI primers (forward and reverse) • 2. Calculate the amount of qPCR reactions required and prepare corresponding volume of the Master mix (amount of reactions + 2) • 3. Pipet the Master mix into the plate (15 ul /well) • 4. Add assigned templates to the Master mix in the wells • 5. Seal the plate with the film. store at 4oC before run • 6. Program the run • 7. Perform the run • 8. Your data file will be uploaded on the course web site • 9. Analyse the data • 10. Write a report • 11. Make a presentation
