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Mastering qPCR
Welcome students!
Welcome! (and contact details)
What is qPCR? (3:09)
Quality control of RNA (quantity, quality and purity)
Assessing RNA quantity and purity with the Nanodrop (11:06)
Assessing RNA quantity with the Qubit (4:12)
Assessing RNA quality (10:11)
End of unit quiz
Reverse transcription
Overview of reverse transcription (9:05)
Selecting the right primers for reverse transcription (6:26)
End of unit quiz
Aspects to consider early on
Intercalating dye or probe-based assays: Which to use? (6:33)
qPCR primer design
Overview of primer design (3:25)
The features of a good qPCR primer pair (8:02)
Designing primers using Primer-BLAST (intercalating dye assays) (11:42)
Preparing new primers for qPCR (3:15)
End of unit quiz
The qPCR run
The difference between technical and biological replicates (2:54)
Setting up a qPCR reaction (10:09)
qPCR plate layout considerations (5:15)
What is a Ct value? (3:01)
What is ROX and should you use it? (5:49)
Melt curve analysis (intercalating dye assays) (4:08)
Optimizing qPCR assays
Workflow overview (3:40)
How to optimize the annealing temperature (6:32)
How to optimize primer concentrations (and the issue of primer dimer) (6:11)
How to optimize other PCR conditions (4:35)
End of unit quiz
Primer efficiencies
Overview of primer efficiencies (6:45)
Calculating primer efficiencies using Microsoft Excel (8:42)
End of unit quiz
Reference/housekeeping genes
What is a reference gene? (5:33)
How to find the best reference gene(s) (7:01)
Downloading and installing NormFinder (MS Excel add-on) (2:12)
Using NormFinder in MS Excel to find the best reference gene (8:22)
Using NormFinder in R to find the best reference gene (4:39)
End of unit quiz
Calculating relative gene expression
Data analysis options (4:26)
The Delta-Delta Ct method (3:45)
Performing the Delta-Delta Ct method using Microsoft Excel (6:12)
The Pfaffl method (2:00)
Performing the Pfaffl method using Microsoft Excel (6:53)
Relative gene expressions with multiple reference genes (Vandesompele method) (2:28)
Performing the Vandesompele method using Microsoft Excel (6:57)
The relative quantities method (2:01)
Performing relative quantities method in Microsoft Excel (3:01)
Always log-transform your data before statistical analysis (6:05)
End of unit quiz
Additional resources
Further recommended reading
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How to find the best reference gene(s)
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