GENET 390 Topic 2 (part 2)
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What is cDNA
Complementary strand of DNA synthesized from an RNA template by the enzyme reverse transcriptase.
What process generates cDNA?
Reverse-Transcriptase PCR (RT-PCR)
What does a cDNA library contain?
A collection of DNA copies made from expressed RNA transcripts (cloned into e-coli plasmids)
What are the advantages to using cDNA
- more stable than RNA
- only contains exons (so it represents splice pattern in tissue/condition)
- gives an expression library
What does Reverse Transcriptase (RT) do?
converts RNA into cDNA
What does a genomic DNA (gDNA) library contain?
DNA sequences representing the entire genome, including coding and non-coding regions.
What is the difference b/w cDNA vs gDNA library in terms of what they represent?
cDNA: only represents expressed sequences
gDNA: represents the entire genome -> including regulatory sequences
Why can cDNA libraries differ between tissues?
Different tissues express different genes.
Why can cDNA libraries differ at different developmental times?
Gene expression changes during development.
Are gDNA libraries expected to differ between tissues of the same organism?
Generally, no. They represent the same genome.
What can cDNA libraries reveal about splicing?
They can reveal different splice variants.
What types of sequences are absent from mature-mRNA-derived cDNA libraries?
Introns and most regulatory sequences, such as promoters.
What is the difference between primers in cDNA libraries vs gDNA libraries?
cDNA: RT uses poly T primers that bind to the mRNA poly A tail
gDNA: random primers
What is the first step in making cDNA?
Isolate total RNA from the cells of interest.
What's the main reason as to why cDNA synthesis differs between eukaryotic and prokaryotic RNA?
Eukaryotic mRNA generally has a poly-A tail; prokaryotic mRNA generally does not.
What is the purpose of a poly-T primer in cDNA synthesis?
It binds to the poly-A tail of eukaryotic mRNA to initiate DNA synthesis.
Why do we also have to add TdT after we add the poly T primer during cDNA synthesis
we need to add a poly-A tail to the newly synthesized cDNA strand, therefore DNA polymerase can make complementary DNA strand (becomes double stranded)
How can we approach choosing a primer when making cDNA from prokaryotic RNA without a poly-A tail?
Use random primers or add a poly-A tail enzymatically (via TdT) and then use poly T primer.
What does cDNA tell us about the genome?
Which genes are expressed and how the genome is being used.
What does PCR stand for?
Polymerase chain reaction.
What does RT-PCR stand for?
Reverse transcription PCR; non-quantitative.
RT-PCR IS _____ & qPCR IS ______.
quantitative or non-quantitative?
RT-PCR: non-quantitative
qPCR: quantitative
What does qPCR stand for?
quantitative [ei Real-time] PCR.
What does qRT-PCR combine?
Reverse transcription and quantitative PCR.
What is the purpose of qPCR?
To quantify the abundance of a specific transcript in an RNA sample.
What is the basic concept of qPCR?
More starting target transcripts generally produce more amplified PCR product.
How can qPCR estimate the original transcript abundance? ei how are we doing the objective
qPCR tracks how much DNA is produced during each PCR cycle to estimate how much RNA was originally present.
Why is comparing multiple transcripts/performing triplicates emphasized in qPCR
measuring gene expression across multiple samples helps assess reproducibility and identify variation between measurements.
Triplicates are particularly useful because RNA is _______and can ______, potentially affecting the results.
delicate, degrade
Why is fluorescence measured during each qPCR cycle?
its how you monitor the accumulation of amplified DNA.
What is a fluor in qPCR?
A fluorescent molecule used to detect PCR product.
What are the two fluorescence-based molecules we use in qPCR that was covered in the slides?
SYBR Green and TaqMan.
When does SYBR Green fluoresce during PCR?
when it binds to dsDNA.
What is the relationship between SYBR Green fluorescence and amounts of dsDNA?
Fluorescence increases with the amount of dsDNA.
What are the 2 cons of SYBR Green?
-It binds any dsDNA, including unwanted primer dimers.
-slowly inhibits PCR rxn
How can primer dimers affect SYBR Green qPCR results?
They can increase fluorescence and overestimate the amount of target product.
Why can SYBR Green fluorescence become less reliable in later PCR cycles?
PCR amplification becomes less exponential, and the reaction can become inhibited or limited.
What is a SYBR Green melt-curve analysis used for?
To check whether primer dimers or non-specific products formed.
How is a melt curve performed?
Gradually heat the PCR sample while measuring fluorescence.
What happens to fluorescence as dsDNA denatures during a melt curve?
Fluorescence decreases as double-stranded DNA separates.
What melt-curve result suggests a specific PCR product?
A single peak, indicating one predominant melting product.
What melt-curve result may suggest primer dimers or multiple products?
Two or more peaks.
What principle does the TaqMan method use?
Fluorescence resonance energy transfer (FRET).
When does TaqMan fluoresce during PCR?
after hydrolysis
What happens when a reporter dye and quencher are close together?
The quencher suppresses the reporter's fluorescence.
What happens when the reporter and quencher separate?
The reporter fluoresces and produces a detectable signal.
Where are the reporter and quencher located in TaqMan?
On the same single-stranded DNA probe.
Why can the TaqMan probe bind the target but not act as a primer?
It lacks a free 3′-OH group needed for DNA polymerase extension.
What happens when DNA polymerase reaches a bound TaqMan probe?
Its 5′-to-3′ exonuclease activity cleaves the probe.
Why does cleavage of a TaqMan probe produce fluorescence?
It separates the reporter dye from the quencher.
What does TaqMan fluorescence indicate?
The accumulation of target PCR product.
What is one advantage of TaqMan over SYBR Green?
It uses a target-specific probe, reducing detection of unrelated dsDNA such as primer dimers.
What does Ct mean in qPCR?
Threshold cycle: the cycle when fluorescence exceeds background and becomes detectable.
What does a low Ct value indicate?
More starting template, because fewer cycles were needed to reach the threshold.
What does a high Ct value generally indicate?
Less starting template, because more cycles were needed to reach the threshold.
Why can replicate qPCR reactions produce different Ct values?
Small differences in starting template or experimental handling can affect results.
What experimental errors can affect qPCR results?
Pipetting errors, inadequate mixing, and RNA degradation by RNases.
Why is qPCR particularly sensitive to experimental error?
The amount of starting template strongly affects the measured result.
What are the two main approaches to qPCR quantification?
Absolute quantification and relative quantification.
What is absolute quantification?
Determining the number of target transcript copies in a sample.
How is a standard curve created for absolute qPCR quantification?
Plot Ct values against the log of known copy numbers from serial dilutions.
What is the purpose of a standard curve?
To estimate the original target copy number from the sample's Ct value.
According to the slides, what copy number corresponds to a Ct of 25 in the example?
1,000 transcript copies in the original RNA sample.
What is relative quantification?
Measuring gene expression relative to reference and control samples.
What two controls are used in relative quantification?
A reference (housekeeping) gene and a control sample, such as untreated cells.
What is a housekeeping gene used for?
Normalizing expression measurements between samples.
What are examples of eukaryotic housekeeping genes in the slides?
GAPDH, actin, and β-tubulin.
Why can choosing a housekeeping gene be more challenging in prokaryotes?
Suitable reference genes are species-dependent.
What is the purpose of comparing treated and untreated samples?
To determine how treatment changes expression of the target gene.
What does the ΔΔCt method estimate?
Relative gene expression changes between experimental and control samples.
What is ΔCt?
The Ct of the gene of interest minus the Ct of the housekeeping gene.
How is ΔΔCt calculated?
ΔCt of the treated sample minus ΔCt of the control sample.
How is fold change calculated using the ΔΔCt method?
2^(−ΔΔCt)
Why are housekeeping genes used in the ΔΔCt method?
To normalize for differences in starting material and sample variation.
What does a fold change greater than 1 indicate?
Higher relative expression in the treated sample than in the control.
What does a fold change less than 1 indicate?
Lower relative expression in the treated sample than in the control.
What is the purpose of a positive control in qPCR?
To confirm that amplification can work.
What is the purpose of a negative control?
To detect contamination.
What does a no-reverse-transcriptase (no-RT) control test for?
Genomic DNA contamination in an RNA-based experiment.
How can primers be designed to help detect genomic DNA contamination?
Design primers that span an exon-exon junction or intron/exon boundary.
Why can genomic DNA produce a longer PCR product than cDNA with intron-spanning primers?
Genomic DNA contains introns, whereas mature-mRNA-derived cDNA does not.
What result is expected if an intron-targeting control primer has no matching sequence in the cDNA?
No PCR product should be produced.
Why is controlling for genomic DNA contamination important in qRT-PCR?
Genomic DNA amplification can be mistaken for transcript-derived cDNA amplification.