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Advanced PCR Techniques Lecture...

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Advanced PCR Techniques Real-time RT-PCR   

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A more accurate method (aka RT-qPCR) In real-time PCR the accumulation of PCR product is measured during the PCR reaction. Reactions include either: - A fluorescent dye that binds to double-stranded DNA - A fluorescent probe that binds to the amplicon Fluorescence is measured after each cycle, allowing product accumulation to be monitored The number of cycles (Cq - the quantification cycle, aka Ct) required to reach a defined, threshold quantity of product is determined - Corresponding to an amount generated while the reaction is still in the exponential phase Higher expression of a gene means a lower number of cycles is needed to reach the threshold

Real Time PCR    

In real-time PCR the accumulation of PCR product is measured during the PCR reaction Determine number of cycles taken to reach threshold amount of product, through real-time measurement of product concentration Tissue 2 has greater expression than tissue 1 - Generates Cq with fewer number of cycles Quantification cycle (Cq) = number of cycles needed to generate threshold amount of product - Lower Cq corresponds to high expression

Performing a Real-Time RT-PCR Analysis    

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Prepare PCR reactions Extract mRNA and covert to cDNA Use primers specific for target gene Primers that are either: - Fluorescent oligonucleotide probe - Fluorescent dye that binds to the amplicon (also need negative control, reference genes etc.) Reactions are commonly assembled in 96 well plates Load into real-time thermal cycler Analysis of the fluorescence of products

Lower Inputs of Template Require More Cycles to Reach the Threshold  

The larger the DNA sample the fewer cycles required to make the threshold Real-time RT-PCR has a large dynamic range (can quantify differences of > 1 million x) compared to end point RT-PCR

Relative Changes in Gene Expression can be Quantified by Comparison of Cq Values     

Real-time PCR was used to compare expression of IL1-b in control eye cells and in eye cells exposed to vitreous humour Numbers show Cq values 11.6 more cycles are needed for control to reach threshold So, control needs to be amplified approx. 211.6 = 3,100x more than treated to generate same quantity So treated cells apparently have 3,100x higher expression of IL1-b than control But: - The reaction may not be 100% efficient, i.e. each cycle may give a less-than 2x increase in product - Quantity & quality of RNA, efficiency of reverse transcriptase reaction etc. may vary between samples - Therefore, one or more uniformly expressed reference genes are needed for normalisation

Reference Gene     

Here’s the same experiment, but now including assays of a reference gene encoding the ribosomal protein RPLP0 For RPLP0, 0.13 more cycles are needed for control to reach threshold So, control needs to be amplified approx. 20.13 = 1.08 x more than treated to generate same quantity Assuming RPLP0 expression is completely unaffected by treatment with vitreous humour, then there must be 1.08 x more high-quality cDNA in the sample from treated cells So, the fold change for IL1-b = 3100/1.08 = 2870x

How Reliable is Real-Time Quantitative PCR? 

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There are concerns re the resolution and consistency of real-time Q-PCR for reasons including: - Variability in the efficiency of reverse transcriptase - Technical issues such as calibration of pipettes - The validity of assumptions made regarding invariant expression of reference genes Biological replicates (different individuals, cultures of cells etc.) and technical replicates (replicate assays of the same sample) are necessary A 2x difference in gene expression can be detected reliably Reported differences below this (and especially below 1.5x) should be treated with caution

Digital PCR      

The most recent innovation in quantification using PCR It allows precise determination DNA (or RNA) concentration A PCR reaction is prepared with a limited amount of template and applied to a silicon wafer - Consisting of thousands of individual reaction wells The reaction contains a fluorescent probe, or a fluorescent dye - Binds to the ds DNA that accumulates during a PCR Some individual wells will receive no molecules of template, some will receive one or more template molecules The PCR is ran and each well scanned for fluorescence

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The number of negative wells, i.e. wells that received no molecules of template, is determined

Digital PCR   

The number of molecules of template applied to the chip can be found precisely using Poisson distribution Mean number of template molecules per well = -log e Fraction of negative wells Example - 8/18 = 0.444 of wells are negative - So mean number of molecules per well = -loge 0.444 = 0.811 - So, concentration of template = 0.881 ÷ volume held in each well...