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To understand the answer to this question, you have to understand what quantitative real time PCR (qRT-PCR) is measuring. Typically qRT-PCR is used to determine changes in gene expression at the mRNA level between different samples. First RNA is extracted from a sample, and reverse transcriptase is used to convert all the RNA to DNA, which is termed complimentary DNA or cDNA. If you start out with a certain amount of mRNA in each sample, the ensuing cDNA will be proportional to it, and will now be compatible with the polymerase chain reaction.
Once you have cDNA, using DNA primers specific to the gene you want to measure, you amplify your gene of interest. Assuming 100% efficiency, each cycle will double the amount of cDNA from the previous cycle. A qRT-PCR machine measures the relative level of cDNA after each cycle by reading the fluorescence from a probe that binds cDNA, and only fluoresces when bound. (There are also other probes but that's another discussion.)
At the end of the reaction, you will have exponential J shaped curves corresponding to each sample that flatten out at the top. If one sample had less of the gene you are measuring, its curve will be farther to the right, indicating that it took longer for the cDNA to amplify to any level (or fluorescence value) on the Y axis. If you draw a horizontal line through the curves in their exponential region, and then solve for the cycle number it took to get to the point where the curve and your line intersect, you will get the crossing point.
The significance of this is that now you have some value by which to compare samples with. If one samples crossing point is 20 and another's is 22, the sample with a crossing point of 20 had 4 times as much cDNA to begin with of the target gene (2^2), or generally 2^dcp (difference in crossing points) gives the relative difference in starting cDNA.
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∙ 11y ago
The crossing point in real-time PCR is the point at which the fluorescence signal of the amplified DNA crosses a predetermined threshold level. It is used to quantify the amount of DNA present in the sample and is used to calculate the cycle threshold (Ct) value. The Ct value is inversely proportional to the amount of target DNA in the sample, with lower Ct values indicating higher amounts of target DNA.
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What is the defference between Real-time PCR and reverse transcriptase PCR?
Real-time PCR is a technique used for quantifying DNA in real-time during the PCR process, while reverse transcriptase PCR (RT-PCR) is used to detect RNA by first converting it to complementary DNA (cDNA) using reverse transcriptase enzyme before proceeding with PCR amplification. Real-time PCR allows for monitoring the amplification process as it occurs, while RT-PCR is specifically used for analyzing RNA levels.
How long has real time pcr been around?
Real-time PCR, also known as quantitative PCR (qPCR), has been around since the mid-1990s. It gained popularity for its ability to monitor the amplification of DNA during the PCR process in real time, providing quantitative data on DNA or RNA targets.
Where can one find an on-line tutorial for Real Time PCR?
You can find online tutorials for Real Time PCR on websites such as Thermo Fisher Scientific, Bio-Rad, or through video platforms like YouTube. These tutorials typically cover topics ranging from experimental design to data analysis in Real Time PCR.
Where online can one learn about real time PCR?
You can learn about real-time PCR on websites such as Thermo Fisher Scientific, Bio-Rad Laboratories, and QIAGEN. These websites offer resources such as tutorials, webinars, application notes, and protocols to help you understand the principles and protocols of real-time PCR.
What is a simple explanation of real time pcr?
Real-time PCR is a technique used to amplify and quantify specific DNA molecules in real time as the reaction is taking place. It measures the amount of DNA present in a sample during each cycle of the PCR process, allowing for accurate and sensitive detection of target DNA sequences. The results are generated in real time, enabling researchers to track the progress of the reaction as it happens.
Differentiate between quantitative and real time PCR?
: Differentiate between quantitative and real time PCR.
What is the defference between Real-time PCR and reverse transcriptase PCR?
Real-time PCR is a technique used for quantifying DNA in real-time during the PCR process, while reverse transcriptase PCR (RT-PCR) is used to detect RNA by first converting it to complementary DNA (cDNA) using reverse transcriptase enzyme before proceeding with PCR amplification. Real-time PCR allows for monitoring the amplification process as it occurs, while RT-PCR is specifically used for analyzing RNA levels.
How long has real time pcr been around?
Real-time PCR, also known as quantitative PCR (qPCR), has been around since the mid-1990s. It gained popularity for its ability to monitor the amplification of DNA during the PCR process in real time, providing quantitative data on DNA or RNA targets.
Where can one find an on-line tutorial for Real Time PCR?
You can find online tutorials for Real Time PCR on websites such as Thermo Fisher Scientific, Bio-Rad, or through video platforms like YouTube. These tutorials typically cover topics ranging from experimental design to data analysis in Real Time PCR.
What are the different types of polymerase chain reaction techniques?
types of pcr: AFLP -PCR. Allele-specific PCR. Alu-PCR. Assembly -PCR. Assemetric -PCR. Colony -PCR. Helicase dependent amplification. Hot start pCR. Inverse -PCR. Insitu -pCR. ISSR-PCR. RT-PCR(REVERSE TARNSCRIPTASE). REAL TIME -PCR
Where online can one learn about real time PCR?
You can learn about real-time PCR on websites such as Thermo Fisher Scientific, Bio-Rad Laboratories, and QIAGEN. These websites offer resources such as tutorials, webinars, application notes, and protocols to help you understand the principles and protocols of real-time PCR.
What are the differences between conventional pcr andreal time pcr?
PCR allows amplification of DNA for a specific gene, after too many cycles of PCR the result will reach saturation, basically meaning all of the DNA has been amplified. Conventional PCR will basically tell you whether or not a gene is expressed in your sample. This can be done semi-quantitavely if the PCR is performed for a low number of cycles, ie it will tell you whether one sample expresses more of your gene of interest than another sample. The results are seen by separating the PCR products by agarose gel/ethidium bromide electrophoresis. Real-time PCR will record exactly what cycle of PCR a detectable level of amplified product became detectable, giving a far more accurately quantifiable estimation of gene expression.
What is a simple explanation of real time pcr?
Real-time PCR is a technique used to amplify and quantify specific DNA molecules in real time as the reaction is taking place. It measures the amount of DNA present in a sample during each cycle of the PCR process, allowing for accurate and sensitive detection of target DNA sequences. The results are generated in real time, enabling researchers to track the progress of the reaction as it happens.
When was Real-time PCR first performed?
The first real-time PCR was first performed in 1993. By 2009, there were seven manufactories that has made as many as eighteen different models. Its a machine that amplifies DNA.
What companies offer real-time PCR data analysis?
Companies which offer real-time PCR data analysis are mainly computer and technology companies like Intel. You could find other companies by researching on-line.
What is a Real Time PCR and what does it offer?
It is a very helpful tool. It is usually used by scientists. They use it to determine something or make certain studies. Real Time PCR offers various effective tools that could be very helpful.
What is a Taqman Real Time PCR?
Taqman Real Time PCR is a technique used in molecular biology to quantify the amount of a specific DNA target present in a sample. It involves the use of specific probes that bind to the target DNA sequence and produce a fluorescent signal during amplification, allowing for real-time detection and quantification of the DNA target. This method is widely used in research, clinical diagnostics, and other applications requiring accurate quantification of DNA.
