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Q-PCR and RT-PCR


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3 replies to this topic

#1 samita

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Posted 25 January 2011 - 02:03 PM

What is difference between semi-qunatative RT-PCR and Q-PCR.

#2 vetticus3

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Posted 26 January 2011 - 09:31 AM

rt pcr is when the products are run out on a gel, and you can see the result as a band. it can't be accurately measured.
q pcr is when a dye is added to the pcr mix, and the amount of dye incorporated into the product is measured by the special pcr machine.

#3 UBClabbie

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Posted 26 January 2011 - 04:38 PM

the above is incorrect.
rt usually refers to reverse transcription. Your template is cDNA reverse transcribed from RNA versus genomic DNA, which is DNA isolated straight from cells or samples.

qPCR does use a dye as stated above sometimes. "q" means quantitative. SybrGreen or TaqMan based methods are used to quantify the levels of template (DNA). Both methods work differently as one is a dye that binds to DNA while the other uses probes linked to fluorescent molecules. Either way, both measure the level of fluorescence. In the case of SybrGreen, the more fluoresence signal over time is a result of DNA amplification. The more starting template, the quicker fluorescence signal will reach a threshold. Semi-quantitative means that you are probably using a comparative method of these thresholds to determine which sample has more target DNA than another. Full quantitative PCR uses a standard to determine more or less exact copy number. So relative versus fully quantitative.

You can have RT-qPCR --> start with RNA, reverse transcribe to make cDNA template, run quantitative PCR to determine relative or exact copy number of target RNA.

I dunno if that was confusing. I tried to condense the ideas....

#4 philman

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Posted 27 January 2011 - 02:34 AM

biotechgirl is correct;
RT-PCR is Reverse Transcription-PCR (i.e. starting from RNA, reverse transcribng to cDNA, then doing the PCR)
qPCR is quantative-PCR, where the amount of DNA after each round of replication is measured, and so you can tell how much DNA was in the sample to start with.

Confusion often arises as qPCR is often nicknamed Real-Time-PCR, which some people (wrongly) abbreviate to RT-PCR, and so get the two techniques confused! Especially as one of the major uses of qPCR is to detect RNA levels within cell types, meaning that many people use RT-qPCR making it even more confusing!




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