RT-PCR and qPCR Flashcards
What does RT-PCR enable?
Enables the study of gene expression by production of cDNA from mRNA using a reverse transcriptase
How is cDNA used?
Used as a template for PCR to detect presence or absence of mRNA of interest
What are the steps of RT-PCR?
- Isolate total RNA from samples
- Reverse transcribe to create cDNA
- Visualise by gel electrophoresis
- PCR-based amplification
What happens to introns?
They are removed
What does the transcript have?
A poly(A) tail
What does reverse transcriptase create?
cDNA:RNA hybrid
Does the forward primer have a template to bind to?
Yes, it has a DNA template to bind to
True or False: Reverse primer can bind to RNA strand
FALSE: it cannot bind to RNA strand
What do oligo(dT) primers bind to?
Poly(A) tail of eukaryotic mRNA
Does mRNA bind to rRNA or tRNA?
No because it is polyadenylated
What do random hexamer primers (RHP) bind to?
Random six-nucleotide sequence
True or false: Compared to PCR, RT-PCR does not require thermocycling
True
What are the two primers RT-PCR require?
oligo(dT)
Random hexamer primers
Why should each sample run a reaction plus a no-RT control?
RNA extraction not always perfect
How is DNA contamination overcome?
Treat extracted RNA with DNase
Use exon-spanning primers
How are primers designed to span?
To span an exon-intron boundary
What happens if primer does not span exon-intron boundary?
DNA not amplified due to no anneal
How are primers designed to flank?
Flank long intron
What happens if primer does not flank?
Short extension time is insufficient for short cDNA sequence
What are one step and two step RT-PCR?
One step - RT and PCR combined
Two step - RT and PCR done seperately
What is endpoint analysis?
Detection of product once PCR has been completed
What does qPCR enable?
Quantitative examination of product levels during course of PCR
What does qPCR analyse and determine?
Analyses exponential phase of product amplification
Determines quantity of target DNA/RNA before PCR
What does qPCR examine?
How many PCR cycles it took to reach threshold that we define
How is qPCR data used?
Set a threshold of certain level of fluorescence/product in exponential phase
What are endogenous reference genes used for?
To standardise results and ensure reproducibility
How are genes expressed in endogenous reference genes?
Expressed at consistent level
What is SYBR Green?
An intercalating dye that binds to dsDNA
Explain SYBR Green?
Amount of DNA/SYBR complex is proportional to amount of PCR product
Only fluoresces when bound to dsDNA
How does one compensate for lack of specificity in SYBR Green?
Can be assessed by melting curve analysis
What is melting curve analysis used for?
To determine specificity of the source of fluorescence and check for primer dimers
What happens when DNA pol. reaches probes of TaqMan?
Forces strand displacement and 5’ exonuclease activity of pol. cleaves the probe so no longer fluoresces
