This video is an easy and full explanation about the principle of real time PCR. For better understanding watch the previous video about the principle of PCR: • The principle of PCR-P...
Good video! just a quick comment to clarify that RT-PCR is Reverse Transcription PCR and Real Time PCR is Q-PCR, for Quantitative PCR, to avoid confusion. Thanks for your video! It was helpful
Her videos are awesome! That's the reason I've put her in my '10 Biomedical Science Channels to watch' video on my own channel! Just a pity she doesn't upload anymore :( Hope for a revival!
I have to admit, pretty impressive ability to comunicate, you really made it cristal clear despite english not being your first language. Thank you very much!
Seriously, your video is totally amazing. What you have shown here, I guarantee you, most people from the field doesn't know. Just one little insignificant correction, cDNA means "complementary DNA" and don't "circle DNA".
I’m actually surprised… I’m first year university student and following the course biotechnology and society in the Netherlands and this came by real fast that I forgot exactly what is was so I looked it up again. I cannot imagine people in the field at least a lot of them not knowing about this if they specialize in this field,
i am not a molecular biologist but i can follow and understand your lecture easily, now i can interpret the COVID19 RT-PCR diagnostic result just looking at the curves and figuring out CT value quite easily, thanks and keep sharing knowledge
Omg thank you soooooo much so much 😭 do you know how many times i tried to understand and looking for videos seeking god to understand this pain in the neck machine?!!! You’re awesome I finally understand it again thank you so much my savior 🥺💕
great video! I already have a Master in Biologie, but never was interested so much in molecular biologie and I totaly get everything about qPCR now! Thanks!!!
This is such a helpful video. Thank you so much for it. The only thing I noticed, was the use of "probability" instead of "possibility" at 17:38, where "probability" means "very likely", in this context. English is weird, sometimes, I know. But right. Your video was extremely helpful and the concept of rtPCR is very interesting. I'm glad it's so simple, too, as I have to be able to do it really soon.
Finally I could understand the key information thanks to this video after days of another mixed incomprehensible video. Please keep doing this. Congr. 👏👏👏
Great video! Thank you, I finally understand this. I have only one question: How the TaqMan hybridize with the DNA at the denaturation temperature and don't "de-hibridize"? or it hybridize at annealing temperature??
Wow started doing qPCR two days ago and I don’t really know what I am doing but this video really clarified a lot. Thanks. You should make a video about analyzing qPCR data using a reference gene along with a target gene. Would really appreciate that!
This video is very good, very absorbing, you explained this methods simply and precisely. The only thing I can say is that SYBR green instead of Cyber green. It's very interesting.
Thank you very much for this video! This is a very clear explanation. One question: could the non exponential plateau phase also due to the fact that all of fluorophore molecules are separated from their quenchers?
Great video! I do have a question, though (and if anyone can answer this, it would be helpful). At 21:20, you mentioned that you include RNAse in the mixture within the PCR tube. I understand that its purpose is to degrade the RNA transcript after it has been reverse transcribed into cDNA, but what is to prevent RNAse from degrading the RNA transcript before reverse transcriptase has had the opportunity to reverse transcribe it?
A correction: the reason of becoming non-exponential phase means there is no more reaction components anymore ( dna pol, dntps etc. run out). The explanation of becoming non-exponential phase is not dna polimerase becomes inactive. By the way, that is a great video, thank you!
dna pol running out? can an enzyme run out that way? my course teacher said two reasons for the non-exponential phase- 1. gradual thermal inactivation of DNA pol after each denaturation step 2. reduced efficiency of primer annealing (due to increasing competition from the template)
Very grateful for your tutorial. I have a little question which has to with your complementary DNA synthesis. I noticed you spoke about use of RNase to degrade the original mRNA from which the complementary DNA was synthesized. I want to know if that's what happens when conducting RT-PCR because I had done cDNA synthesis where the the template strand was RNA and the nascent strand was DNA. So please clarify that for me. Thanks
Hi! Thanks for the video, the explanation is really good. I have one question about the graphs at the end. You told that some lines meet the thresold line before than other because in the beginning some samples have more DNA copy than others. I don't understan very well why this happen, is it for different concentration of the sample, kinetik or something else? Thanks.
I dnt know how to say thank you.... i have no words.....😥😥😥 you are amazing...and this is a wonderful video, i watched more videos of you, thank you sooo much n god bless you♥️♥️♥️
Thank you so much miss for a such a useful and simple explanation. 15:34 you mentioned the annealing and extension occur in 3 to 5' direction while hitting the fluorescence. I think this process is from 5 to 3" but tell us about the position of the probe in case of 5 to 3. thank you so much we really appreciate your work mam. This is the first video I have seen with such an amazing animated explanation.
thank you for this explanation that was great yea i have some difficulty when i want to know the meaning and for what we can use the the real time pcr analysis section like RN delt rn and RQ ...... so on
Nicely explained. Can you please tell me where exactly to set the ct value while analysing say 96 samples in multiplex? Since on diagnosis we normally do not go for replicates and therefore it becomes difficult and at times confusing where exactly the thresh hold be set.
Thanks a lot for your video!!! Just to understand more about the housekeeping gene, using SYBR green, for example..the primers of GAPDH go together with my primers of interested in my mix? in that way, I will have a duplex PCR, or I need to do another reaction? Thanks a lot for your time! ;)
Thank you for your video! I have a question: what is the value of the threshold line dependent on? The detector we're using? The Fluorometer? I ask because I'm not sure, for example, if I analyze gene expression of let's say, 6 different genes (6 different TaqMans), whether I'll be able to compare their threshold lines and make assumptions about one gene's expression levels in relation to the others.
Normally, when we apply the rt-PCR we set the machine, so we choose the number of samples and the fluorophore we are using, and the machine automatically chooses the threshold. Furthermore, rt PCR is usually performed to compare the expression of the same gene in different cells. On the other hand, what are you talking about is a special case called (Multiplex rt-PCR) (see 18.06 min) which is to compare the expression of different genes in the same experiment (to use different taqmans).
Hello and congratulations on your videos. I am very pleased with the amount of detail you were able to provide in such short time. Do you have plans to make a similar video about digital PCR? Greetings
Thanks for the video, I have several questions - I just got a job in the Molecular Hematology lab and needs to learn PCRs in details. Why would someone wants to do real time PCR, like why do you need to know how much DNA is made at a particular time, wouldn't the final measure after - say an hour would be sufficient? So the purpose of the reverse transcription PCR is to measure gene expression. The reason you do RT rather than just PCR is because you want to measure the mRNA (post transcription) rather than DNA (pre transcription - which doesn't tell you if the gene will be transcribed or not), right? Lastly, my lab is really concern about contamination. They even have separate room for Pre-PCR and Post-PCR. They do not allow anything, and I mean ANYTHING, including cell phone or pipette tips from the Post go to the Pre room. Like it can contaminate through air or something? I've never done any PCR before, all I learn is from textbooks.
thank you very much for what you are doing ! i love how you take your time to explain well i do have a request video if you please, can you take some of your time to explain the ELISpot technique (enzyme-linked immunospot) thanks
Just to clarify or maybe mention that the probe can be specific or generic and the same with the primer it can be specific or generic making of a robust primer/probe design combination. I think I am a little confused on when the probe will hybridize to the genomic DNA. Does the taqman probe hybridize to the source (genomic DNA) at the first cycle or does this occur after several cycles. I would think there would be many mistakes (false reactions) if the Probe Hybridizes to the un-amplified DNA (before the first cycle with primers) rather than hybridizing to the DNA fragment after several cycles. If this cannot be controlled in any way then this would a rather large disadvantage of the taqman assay giving more false positive results. Let me know what you think about that.
