qPCR DATA ANALYSIS | REAL TIME PCR скачать в хорошем качестве

qPCR DATA ANALYSIS | REAL TIME PCR

Quantitative PCR, Real-time PCR or qPCR as it is known, is similar to traditional PCR in that you require primers for the target region you wish amplify. As with traditional PCR, you are aiming to make multiple copies of the region of interest via the designed primers. Where do you get these primers from? You obtain the sequence of the gene of interest via a cite such as the National Center for Biotechnology Information (NCBI) and identify the region you wish to amplify. A reminder that the primers are designed so that you make numerous copies of that region, relative to the rest of the other genes in the chromosomes of the genome. THe primers you design flank the forward and reverse region you are interested in. With traditional PCR, you get a result of whether the region you targeted was present in the genome, at the end of the complete reaction (we’ll talk about the number of cycles you generally use to ensure adequate copies of hte gene of interest is created - if present in the sample, shortly). qPCR adds an additional apportunity for information by including either double-stranded DNA binding dyes or specific fluorescent probes, such as Taqman to report how much of your region of interest is present at each cycle of the PCR reaction, not just at the end of the complete reaction. Why might you want this kind of information? For certain situation, example, viral infections, you don’t just want to know whether a virus is present in the cells of a sample, but you want to know how much of the virus may be present. In certain diseases, this may be critical in determining whether treatment is needed or recommended. Now what happens with the additional fluorescent molecules or probes that are incorporated during a qPCR, is that the fluorescent signal intensity increases from each cycle of the qPCR reaction if the gene you are targeting is present. This is a result of the doubling of the gene template from cycle to cycle. In the case of where a specific probe is used in addition to the primer, the probe has a molecule - called a quencher - that stops the fluorescent molecule from giving a signal if it is not specifically bound to the region you are amplifying. Hence with qPCR, if using a specific probe fluorophore, you design not just forward and reverse primers, but primers unto which a fluorescent molecule and quencher is conjugated. As you can appreciate, this gives you a second layer of specificity. Of course, you could opt for an easier but potentially less specific version by using a dye to binds any ds DNA product. Because you have the additional of fluorescent molecules, you need a PCR machine that has lasers to supply energy that excites the fluorescent molecules, and detectors and record the fluorescent wavelength emitted in response. Take home msg: you cannot do qPCR on a regular or traditional PCR machine. Now I keep talking about cycles of the PCR, and the fluorescent signal being detected at the end of each cycle rather than at the end of the complete PCR reaction. So let’s look at how PCRs operate: You first denature the ds DNA templates that form the genome of the cells or biological samples you are analysing. The primers bind to the region that have been designed to bind to on the DNA templates. By repeating the DNA denaturation and primer binding cycles (called annealing) for multiple times, you copy the region you have designed the primers for: an analogy here is like when you’re photocopying and you change the number of copies to how ever many you need. On a practical and real lab side, you will include standards. Where you need actual quantities for particular diagnostic or disease management decisions, after confirming that the reaction as proceeded as appropriate: you may determine the viral load for the reported Ct/Cq value. This is done by dividing the starting quantity of the viral gene you’ve detected - based off the standard curve by the starting quantity of a control gene that you’ve tracked and know it’s expression to be stable. . . Animation credit: Adapted from New England Biolab . . . // ADWOA I'm Adwoa (Adwoa Biotech), a Biotechnology graduate. I’ve worked in medical research for years and want to be useful to people new to the lab life. This channel takes you through some of the techniques and concepts I've learnt working as a Research Assistant. Hopefully it helps if you're new to the topic/technique. . . This video is about quantitative pcr, quantitative PCR analysis, quantitative pcr technique, quantitative PCR explained, quantitative PCR vs real time PCR, quantitative PCR protocol, quantitative pcr vs regular pcr, quantitative, real time pcr, retime pcr procedure, real time pcr animation, real time pcr test, real time pcr experiment, qpcr, qpcr explained, qpcr data analysis, qpcr protocol, qpcr analysis, qpcr basics, qpcr principle, qpcr standard curve, qpcr technique, qpcr taqman