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!
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
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!
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)
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 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.
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.
The doctor said that my parent's CT value needs to be at certain number for them to be able to leave the hospital. I have no idea what it was all about. I came here without any medical knowledge, but your explanation in the video is very good and clear. I don't really understand the biological details but i get the concept of how it works. Thankyou thankyou!!!
CT value is the threshold value of fluorescence emitted. More than the CT value means the "gene of interest" (pathogenic gene of virus) has been ascertained. If it's less or nil, then the individual may clinically be diagnosed as "free of the viral infection/disease "
Finally I could understand the key information thanks to this video after days of another mixed incomprehensible video. Please keep doing this. Congr. 👏👏👏
an important component for the reaction preparation was missing. The salt buffer. Without optimal pH-value and corresponding divalent cations the polymerase would not work properly at all
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.
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!
Watched this with my girlfriend, thoroughly enjoyed it. She's doing PhD genetics, I was a biochemist 15 years ago. Can I just add some constructive criticism on the language clarity front: "quencher" is pronounced "kwencher", as in "to quench", like when you're thirsty. As an Englishman, I feel I should apologise for our impossible language, your English is very very good!! Also, parent ssDNA is not copied by Taq polymerase/TaqMan, it is extended. The slide makes sense, just not the word "copy" in this context. Keep up the fantastic educational work! I learned something today :)
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♥️♥️♥️
Your lecture clarifies many doubts and basics of PCR for me. I highly obliged to your efforts. Still, I have couple of queries. 1. What is actual meaning of Ct value and delta Ct? 2. Many a time, Why we use RNA sample to synthesize c-DNA instead of direct DNA sample for RT-PCR?
For your second query, for instance in case of covid 19 virus detection we have to use reverse transcriptase pcr because it is a rna virus. So it all depends on what type of nucleic acid you won't to detect
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.
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 really helpful video but I think that it is SYBR Green not Cyber green, it really helps me to clarify some doubts that I had thank you so much, by the last qPCR: IS REAL-TIME PCR and RT-PCR: is REVERSE TRANSCRIPTION PCR.
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?
Amazing! I am from Brazil and your explanation does an accessible language (I do not speak English very well). Congratulations on your class. (I use google translate legend kkkk)
Amazing video. It is very detailed. was an undergrad, i didnot know the difference between cyber green and taqman. And then in conferences, I didnot understand why Ct in everyone's y axis.
I'm not sure if you already figured out the answer, but if not here's an explanation: The SYBR Green that is added to these reactions is very saturating, in other words, a ton of it is added. Thus, SYBR Green DOES bind to every minor groove in all DNA in the sample until there are tons of copies of DNA. Now, you may ask, when there are tons of copies of DNA, what happens to SYBR green dye binding? At that point, there is not enough SYBR Green in the solution to bind to ALL the minor grooves in the DNA, and there is variability. However... Why doesn't this variability when SYBR Green affect your results? Take a look at the threshold line, which is discussed at around 24:55 in the video. The threshold where the Ct value is calculated will be very very low on the amplification curve, where you can be sure the SYBR green dye is still completely saturating and binding to all the minor grooves on all the DNA. This threshold Ct value is what is used in relevant calculations for gene expression, so that is why we do not need to worry about SYBR Green running out and not binding to all the minor grooves. Another point to keep in mind here is that RT-PCR is all relative, so you can't compare between two different sequences because there are many variables besides just how much SYBR binds, such as efficiency of a primer set for which you may not have information/data. In other words, the idea is that with the same reagents, the same amount of dye will always bind to the same amplified fragment of DNA, it doesn't matter if that's 50 or 500 molecules of dye.
Nice one! Can you please explain about how the melt curve looks like for NTC? and How Ct value is used to determine the amount of gene vis-a-vis housekeeping gene
The earlier the CT is (the earlier the amplification curve crosses the threshold line), the larger is the amount of gene at the beginning of the experiment ... you do not compare the gene of interest with the housekeeping gene, you compare the CT of the gene of interest in different samples. The housekeeping gene is a gene that is expressed similarly in all the samples so we are sure that all the samples exist in the same amount. In other words, the CT of the housekeeping gene should be the same in all the samples, otherwise, your experiment is not precise. I hope I answered your question ...
great video. At around 27 mins you attribute the 'plateau' phase to decreased activity of the polymerase. Isn't this plateau effect just a result of the dntps in the mixture being exhausted?
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??
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
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).
Very good explanation of the process in general. Just a comment that the plateau phase or non-exponential phase is not due to inactivation of DNA polymerase but due to the concentration of the fluorescent dye reaching beyond maximum detection limit. Say for example, you can lit a dark room, and the light will illuminate the room. Keep on increasing the number of lights, and the room will glow more and more until it will reach a saturation point beyond which no increase in light intensity will be visible even by doubling the number of lights. So if the room reaches at full saturation point with 50 lights, increasing the number of lights to 100 or 500 will have no more effects.
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!
Good explanation
Biomed Master yeah but small blunders.....she need to improve that......and she’ll become best
Intetesting!
" RT-PCR is Reverse Transcription PCR" That's what I thought. She says "Real Time" So what's the difference?
I came here crying due to I couldn't understand my class today. I left loving RT PCR. Amazing work! Thank you so so muuuuch!
Thank you for your comment, it makes me happy o hear that :)
Yes, Manuel, very handy and great video! Will help our QFT team for sure
@@biomedicalandbiologicalsci4989 Egyptians are making good videos.
@@biomedicalandbiologicalsci4989 amazing. Your videos still having big impact.
This 5 years ago video has provided me with great knowledge and insight. Thank you very much for the clear and detailed explanation
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".
She said cyclic not circle
@@mehwisha.salman5517 still wrong
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,
It is an amazing video! thanks a lot for this explanation. Just one correction; cDNA is for "complementary" not for "cyclic" (22:04). Great job!
This is the most logical progression for someone who's looking to understand PCR. Really well structured content, big up!
Good presentation .. Just an important point: cDNA is Not cyclic DNA, It's Called Complementary DNA.
Thanks for correction
Yes👍
Good presentation
@@boinualizeh4877 good
Means this cDNA undergoes this cyclic reaction so one can say.
your channel deserves more views! i could not have done well at university without you
Thank youu .. this really makes me happy :)
@@biomedicalandbiologicalsci4989 Hello Madam, Why have you stopped making videos? Continue please
This is absolutely incredible. Just what I needed for a presentation next week.
HAHAHA, very good. Hope you put my channel as a reference :p
Biomedical and Biological Sciences I hope too! Very good informations in you video!
@@biomedicalandbiologicalsci4989 can i have this presentation as a file?
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
U explain well. I didn't understand RT PCR for my course. Now I understand it. U have great teaching talent.
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!
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)
You are magnificent.
You do not leave any point without detailed explanation.
God blessings ❤
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 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.
all mechanisms explained clearly! no insignificant details, just the main points, thank you : )
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.
Thank you madam for your awesome explanation....first I didn't understand the rt PCR before now I'm clear in this topic ...once again thank you madam
Haven't seen such a complete and educative video!!! Thank you.
I wish you had more views. You are such a good lecturer and I am really grateful for your time in uploading these videos!
The doctor said that my parent's CT value needs to be at certain number for them to be able to leave the hospital. I have no idea what it was all about.
I came here without any medical knowledge, but your explanation in the video is very good and clear. I don't really understand the biological details but i get the concept of how it works. Thankyou thankyou!!!
CT value is the threshold value of fluorescence emitted. More than the CT value means the "gene of interest" (pathogenic gene of virus) has been ascertained. If it's less or nil, then the individual may clinically be diagnosed as "free of the viral infection/disease "
Finally I could understand the key information thanks to this video after days of another mixed incomprehensible video. Please keep doing this. Congr. 👏👏👏
You are an excellent tutor, very clear message. I learnt things I could not understand from other videos.
Your explanation is so succinct and understandable even for a non-scientist like me!
an important component for the reaction preparation was missing. The salt buffer. Without optimal pH-value and corresponding divalent cations the polymerase would not work properly at all
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! I needed to refresh this topic, and you covered it all, good job!
Awesome. Your presentation and explanation are clear. The best lecture out there. Keep your lectures coming.
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!
Watched this with my girlfriend, thoroughly enjoyed it. She's doing PhD genetics, I was a biochemist 15 years ago. Can I just add some constructive criticism on the language clarity front: "quencher" is pronounced "kwencher", as in "to quench", like when you're thirsty. As an Englishman, I feel I should apologise for our impossible language, your English is very very good!! Also, parent ssDNA is not copied by Taq polymerase/TaqMan, it is extended. The slide makes sense, just not the word "copy" in this context.
Keep up the fantastic educational work! I learned something today :)
Its your* English. Not "you're" English :)
@@drmarium correct. It's also "it's", not possessive "its". :)
this is the first time i got what rt-pcr is.thanks a lot
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♥️♥️♥️
This video provides an easy way to understand RT-PCR and real time PCR. Thanks! Hope you can provide more video, and many people can access this page.
Your lecture clarifies many doubts and basics of PCR for me. I highly obliged to your efforts. Still, I have couple of queries.
1. What is actual meaning of Ct value and delta Ct?
2. Many a time, Why we use RNA sample to synthesize c-DNA instead of direct DNA sample for RT-PCR?
For your second query, for instance in case of covid 19 virus detection we have to use reverse transcriptase pcr because it is a rna virus. So it all depends on what type of nucleic acid you won't to detect
This what i am looking for. Thank you for your effort and nice presentation...
Well done. The best explanation I could find!
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
It is a nice video with a great explanation.
It is helpful to me
More Useful video for Molecular biochemistry Research work...Thank you mam for your video ..
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?
here we are using RNase H it cleaves only the RNA-DNA hybrid then you get cDNA right
Nice effort in giving information to the beginners and even the experts. Thanks for the video
A really helpful video but I think that it is SYBR Green not Cyber green, it really helps me to clarify some doubts that I had thank you so much, by the last qPCR: IS REAL-TIME PCR and RT-PCR: is REVERSE TRANSCRIPTION PCR.
Thank you for the very informative video. Easy to understand and to listen to your English accent. and very great presentation skills. Thanks a lot
Clear and simple explanation. Thank you and keep up the great videos.
You are amazing it took me years to understand ft pcr. But you did it in few minutes
Thank you very much for this video ! You explian very clear and easy to follow. Hope you keep doing this
Hi! I’m so proud of myself because I understand that your explain! Very very thank you!😂😂😂
The way u try to make us understand is just lovely.. thanks for the video and explaination 🙏🙏❤️
Thank you so much!!! it´s a really good explanation :)
Thank you for the nice comment :)
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?
Thank you. Your explanation is very clear and the slides you made are awesome.
Very good video you did an amazing job simple and doesn’t miss important details, but doesn’t go into an unnecessary amount of detail.
Should we tell her it's SYBR green not Cyber green?
Indeed bro.
cyber green sounds cooler.
Yes
also cDNA it’s a complementary DNA not cyclic DNA
Clarity in ur presentation. I enjoyed it.
Thank you very much
Today I understood real time PCR ( nice and simplified explanation) thank you so much
Nicely Explained, thanks!!
Amazing! I am from Brazil and your explanation does an accessible language (I do not speak English very well). Congratulations on your class. (I use google translate legend kkkk)
Amazing video. It is very detailed. was an undergrad, i didnot know the difference between cyber green and taqman. And then in conferences, I didnot understand why Ct in everyone's y axis.
Very best explanation of the PCR tech I have ever seen. Thanks!
This is Apt and Well Explained. Many Thanks.
Question: What if cyber green attaches to more than one minor groove of a DNA copy? Or is that not possible? How?
I'm not sure if you already figured out the answer, but if not here's an explanation:
The SYBR Green that is added to these reactions is very saturating, in other words, a ton of it is added. Thus, SYBR Green DOES bind to every minor groove in all DNA in the sample until there are tons of copies of DNA. Now, you may ask, when there are tons of copies of DNA, what happens to SYBR green dye binding? At that point, there is not enough SYBR Green in the solution to bind to ALL the minor grooves in the DNA, and there is variability. However...
Why doesn't this variability when SYBR Green affect your results? Take a look at the threshold line, which is discussed at around 24:55 in the video. The threshold where the Ct value is calculated will be very very low on the amplification curve, where you can be sure the SYBR green dye is still completely saturating and binding to all the minor grooves on all the DNA. This threshold Ct value is what is used in relevant calculations for gene expression, so that is why we do not need to worry about SYBR Green running out and not binding to all the minor grooves.
Another point to keep in mind here is that RT-PCR is all relative, so you can't compare between two different sequences because there are many variables besides just how much SYBR binds, such as efficiency of a primer set for which you may not have information/data. In other words, the idea is that with the same reagents, the same amount of dye will always bind to the same amplified fragment of DNA, it doesn't matter if that's 50 or 500 molecules of dye.
@@hgupta32323 I was having the same thoughts as well. Thanks for the answer!
Thank you for this amazing video , really i gain great knowledge about qpcr this due to you amazing lecture , i am looking forward to more lessons
Nice one! Can you please explain about how the melt curve looks like for NTC? and How Ct value is used to determine the amount of gene vis-a-vis housekeeping gene
The earlier the CT is (the earlier the amplification curve crosses the threshold line), the larger is the amount of gene at the beginning of the experiment ... you do not compare the gene of interest with the housekeeping gene, you compare the CT of the gene of interest in different samples. The housekeeping gene is a gene that is expressed similarly in all the samples so we are sure that all the samples exist in the same amount. In other words, the CT of the housekeeping gene should be the same in all the samples, otherwise, your experiment is not precise. I hope I answered your question ...
This was such a life saver 🤧🤧❤❤❤🤧🤧
What is the different between Cq and Ct
This is such an informative video! Very useful for a graduate course I'm considering reattempting. Cheers
well explained in the simplest form ever! Kudos
Another wonderful, informative and concise video, as always. Keep going
Outstanding job very informative and easy explanation of difficult thoughts many thanks for your knowledge sharing
Really helpful for refreshing the whole thing... Thank u so much...
Thank you for such a simple and clear explanation between these two assays.
Very well explained, will be helpfull to understand the basics of RT PCR, Thank you
Really awesome video. Super helpful in clarifying this concept for me. Thank you!
Great work :) We need some videos about Flow Cytometer :)
Your step by step explanation is excellent
Thank you
Great explanation... And significant points covered... Keep uploading such videos.. looking forward to it...
Thank you so much, it helps me alot to understand about pcr.. You explained it well so it easier to understand 👍
Your method of teaching is very good...please give a lecture on somaclonal variation
great video. At around 27 mins you attribute the 'plateau' phase to decreased activity of the polymerase. Isn't this plateau effect just a result of the dntps in the mixture being exhausted?
This video clarified a lot.. Thanks 🤩
no doubt its an excellent presentation about RT-PCR
Excellent depth of knowledge presented simply
She explained the concept of quantitative pcr towards the end of her lecture to avoid confusion.
This helped it finally click for me! Much appreciated.
Thank you so much. The presentation was so clear. Keep up the good work.
It was really very beautiful video and also your accent was so nice. Thanks a lot.
You explain it well friend. Thank you so much.
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??
Hello ! Thank you for this clear video. At 22:01, cDNA it's not cyclic DNA but complementary DNA, a DNA without introns.
At 27:57 All dNTPs are consommed.... Maybe the polymerase is not efficient as the beginnin but it's not the most likely explanation..
Good Job. The Detector name is SYBR green, not Cyber Green
Thanks for such a nice explanation, it was really helpful. Please make more videos like this.
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
You are the Bioanalysis queen!
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).
Thank you so so much ma'am, this video helped me alot.♥️
Very useful video, thanks alot 👌👌👌
Very good explanation of the process in general. Just a comment that the plateau phase or non-exponential phase is not due to inactivation of DNA polymerase but due to the concentration of the fluorescent dye reaching beyond maximum detection limit. Say for example, you can lit a dark room, and the light will illuminate the room. Keep on increasing the number of lights, and the room will glow more and more until it will reach a saturation point beyond which no increase in light intensity will be visible even by doubling the number of lights. So if the room reaches at full saturation point with 50 lights, increasing the number of lights to 100 or 500 will have no more effects.
thank you very much. i like your explanation. best wishes from Turkey :)
Great simplified explanation...Thank you
welcome :)