Quantitative real time PCR (qPCR)

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A quantitative polymerase chain reaction (qPCR), also called real-time polymerase chain reaction, is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR), which is used to amplify and simultaneously quantify a targeted DNA molecule. For one or more specific sequences in a DNA sample, quantitative PCR enables both detection and quantification. The quantity can be either an absolute number of copies or a relative amount when normalized to DNA input or additional normalizing genes.

The procedure follows the general principle of polymerase chain reaction; its key feature is that the amplified DNA is detected as the reaction progresses in "real time". This is a new approach compared to standard PCR, where the product of the reaction is detected at its end. Two common methods for the detection of products in quantitative PCR are: (1) non-specific fluorescent dyes that intercalate with any double-stranded DNA, and (2) sequence-specific DNA probes consisting of oligonucleotides that are labelled with a fluorescent reporter which permits detection only after hybridization of the probe with its complementary sequence to quantify messenger RNA (mRNA) and non-coding RNA in cells or tissues.

qPCR is the abbreviation used for quantitative PCR (real-time PCR).[1] Real-time reverse-transcription PCR is often denoted as: qRT-PCR[2][3][4] The acronym "RT-PCR" commonly denotes reverse transcription polymerase chain reaction and not real-time PCR, but not all authors adhere to this convention. Source of the article published in description is Wikipedia. I am sharing their material. Copyright by original content developers of Wikipedia.
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Sir, you're blessing for us (students). The way you explain everything, on point, that is needed, is really appreciable! Can't Thank-you enough Sir. 🙏🙏

anjalibahot
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the phases described here is wrong. Phases includes 1. baseline 2. exponential 3. linear and 4. plateau. Here baseline is described as exponential, exponential as linear, linear as plateau.

joydeepsen
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At 9:15 u could've used the phrase, "in real time", to make sense of its name, good explanation :)

adarshguptak
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On which ct value we can say test is positive or negative?

hmh
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Dr.Bhattacharjee, thanks for the post. Its really helpful. I am a physician, and I have zero experience on PCR.  We ran a real time PCR on fecal sample for analyzing gut microbiota (i.e., in a given fecal sample, how much bacteroids/lactobacilli are present?).  Is it feasible to post a video on analyzing the data? I would really appreciate it.

Thank you

kumarsvc
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Hello Shomu,
I am struggling with the Ct values I got from my lab.
For my gene of interest I got no amplification for the negative control cells, for the test cells I got 30.935 and control cells 33.759. For my reference gene I got 30.310 for negative control, 28.260 for test and 28.022 for control. I got wrong results since the negative control where there should not be amplification and the ct values are too high. However I need to calculate the delta ct for test (I got 2.67) and for control (I got 5.7) to compare house keeping and gene of interest. But what do these values really tell me? And for the double delta Ct I got 8. What does it mean?
I hope to have a reply from you soon!!
Best wishes,
Natalie

sdikwella
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Thank you so much Dr. Bhattacharjee! For me it is my first lecture to study qPCR. It was clear and useful to understand. I have question about melting point. If I want to know the size of amplicons that has melting point around 88C, how can I know?

nahyuncho
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Shyam has 5 treatment experiment so how many times he has collect the samples and how many times he has prepare cDNA?

mlawandepatil
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What is the difference between semiquantitative and quantitative pcr?

sheelachoudhary
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RNA quantitative target not detected in lal path lab neg or pos

rajarora
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Hello, would you mind telling me where you got the graphs from? Thanks

katiecablankdaboom
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Please sir make the video on RT LAMP method

ishmitalohani
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"It's a bonanza for us."

veryirie
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your explanation was so much complicated ....very bad

shajidislam