thank you SO much. i was struggling to understand amidst so much stress but this completely cleared up my confusion and i could not be more grateful right now
This is a quite old comment I know, but wanted to let you know that it's comforting to see that even a master's student needs clarification on this. I'm doing my bachelor's and currently battling imposter syndrome, so thank you.
@@mariemasarikova8575 Haha, thanks for clarifing my syndrome= imposter syndrome-" It disproportionately affects high-achieving people, who find it difficult to accept their accomplishments" I thought i was able to find cure for HIV :D
Nah it's just that people subscribe for so many different subjects, any given video will only appeal to a small percentage of them. But spread the word!
Sir but how can this be used for quantitative assay of a particular gene...i mean i only understand that this will be only useful for qualitative analysis
the general idea: with each pcr cycle, the amount of a certain DNA sequence is double. Using some kind of labeling, e.g. fluorescent dyes, we can detect the presence of the mentioned DNA sequcense. The fluorescent dye has a detection limit (minimal concentration of the substance which can be detected), so, if we know that we detected the DNA after, let's say, 9 PCR cycles, and the detection limit for the dye is 100 nmol/ml, that means that the amount of DNA in the initial sample was 2^9 times lower, thus, quantitative analysis.
Hey Dave, just a quick question. How would the cDNA bind to the DNA in microarray if the primary transcript formed after trancsription is spliced? you can just link me some resources for the answer if you want to because i have been watching your videos and you seem to have quite a busy schedule.
C DNA binds to DNA in microarray due to phenomenon called complimentary base paring, or we can say that A base always bind to T like that all base pairs are bound to attach to their complimentary strand due to all complimentary bsepairs . If i am wrong correct me.
@@shrikantnalkar4751 no i get that... i am aware of complementary base pairing.. my question was if the transcript of DNA goes under splicing, how does the cDNA bind to oligonucleotides on microarray... turns out oligonucleotides are the mRNA from which the cDNA is made and not a piece of DNA in the microarray... basically my doubt is solved after doing some research but thnx anyway
I just wanted to give a heartfelt thank you for this video. My first year final tomorrow is an essay about using microarrays to identify heterochromality genes in huskys and this helped me study for it tremendously.
During the process of RT-PCR we get a complimentary ssDNA for which we perform PCR to get a dsDNA, we add this to each of the wells of the array plate and the complimentary DNA probe hybridizes and form a link. My doubt is if the DNA is already ds post RT-PCR how will it bind to another DNA probe. Or are we considering the DNA to be ss post RT- PCR. (the animation does not support the fact that the DNA is ss post RT-PCR at 5:11 ) Anybody?
Why getting a 90€ Biochemistry-Textbook when RUclips can help you to understand the topic in about 8min? :) Thank you for your content! It helped me a lot! :)
Dude seriously. I'm learning Biochemistry and required to use the Lehninger book that everyone says is so good but I honestly am learning so much more from RUclips and only refer to the book when I have to. I'm getting along just fine without it.
Thank you so much for the video! I have a question. Does the cDNA have to be denatured, like with NaOH or something, so that it can bind to the single-stranded probes in the array?
I'm a little confused that the approach is to make double-stranded DNA with DNA polymerase and then try and get it to apparently separate and bind to single-stranded probes. Do the probes test for both the sense and antisense strands on each spot?
Hey, you might already have found the answer to your question but in order to make cDNA we use a reverse transcriptase. This is a protein that basically turns your RNA back into DNA. Usually the reverse transcriptase leaves you with a single stranded DNA strand and a round of normal PCR (polymerase chain reaction) is required to turn it into double stranded DNA. Hybridization can then be performed by separating the strands again (either heating or chemical, where heating would be preferred since it is less harmful for the DNA). The stand that is complementary to the probe that was designed will then bind to either one of the strands and start the microarray assay. Which strand binds should not matter too much since the probe should be specific for a certain gene. Hope this helps.
This is great: super simple, super precise, it makes you really understand the basis of microarrays. From here you can go to more complex videos, but you do need a basis like this to start! Congratulations
So for microarray, we are only using one cDNA strand which was made from the template, the mRNA. However, there is one thing that isn't clear to me and that is about the RT-PCR. RT-PCR generates double-stranded cDNA right? How come in microarray we are only using the single strand and not double? Is there any intermediate enzyme that was involved?
In a nutshell, RNA splicing involves the removal of introns (non-coding parts of the mRNA) and joins the coding sequences (exons) together in order to enable translation. Since the parts removed are non-coding, it does not affect RT-PCR. RT-PCR, standing for Reverse Transcription Polymerase Chain Reaction, involves reverse transcriptase - converting RNA into cDNA - and (often) Taq DNA polymerase - completing the DNA after denaturation. To sum up, since the RT-PCR process only duplicates CODING DNA, DNA splicing does not affect the process AT ALL, since it removes NON-CODING parts of the original DNA.
DUMB QUESTION: why can't the original DNA with the gene be isolated from the beginning? Why must the mRNA be isolated first, just to then be reverse engineered into the original DNA?
The way I understand it, not all of the genes present in the DNA, are being expressed/transcribed. So in order to only identify the currently transcribed genes, you have to work with mRNA.
word of caution, rna pol doesnt need a primer right, so reverse transcriptase having a primer feels fishy but its not because although widely called reverse trasncriptase the enzyme itself is synthesizing dna and so in essence its a dna pol and dna pols need the TTT primers.
I wish you could see what im seeing people! I wish you could be here with me!!! oooohh Baby HAHA! BRING IT OOOONNN! AND REMEMBER FOLKS! YOU HEARD IT FIRST FROM JOHNYYYYAAAHHHHAHA! *dead*
@5:26 Do I have this right? (no background in science, just leaping in) -There's a double stranded cDNA: one is identical to the DNA template and the other is complementary to template --These two strands are bound together, right? -"One of the two strands of some cDNA molecule made from an mRNA ought to bind to the DNA fragment in a particular well" --How does one of the strand bind to the DNA fragment? My assumption is that the cDNA are bound to each other, so how does one of them separate and bind to the fragment?
thank you SO much. i was struggling to understand amidst so much stress but this completely cleared up my confusion and i could not be more grateful right now
Currently, using this to supplement my Master's program. The visuals helped to solidify my readings. Thanks, Dave!
This is a quite old comment I know, but wanted to let you know that it's comforting to see that even a master's student needs clarification on this. I'm doing my bachelor's and currently battling imposter syndrome, so thank you.
@@mariemasarikova8575 I’m doing this as a 16 year old , SED
@@mariemasarikova8575 Haha, thanks for clarifing my syndrome= imposter syndrome-" It disproportionately affects high-achieving people, who find it difficult to accept their accomplishments"
I thought i was able to find cure for HIV :D
you just nailed it🔥 love from India💝
Huge thanks jumping from Saudi to you and the channel!
I'm doing a biochemistry masters and this is helping me - thanks.
Plz make video on validation and QC of flowcytometry and molecular techniques like RFLP ,SSP and SSOP
this man is a legend i have an assignment due tmrow that if i fail im gonna have a 70 and this man just saved me
Thank you sir very beautifully you explain the procedure
thank you for your content. you explained it beautifully
thank you
Tku sir I wish I could have been your student.
Thankyou sir ✨
Wow, nice video man
Good work
Is it me or does it seem like Dave's videos are being suppressed the amount of views to subscribers just does not add up
Nah it's just that people subscribe for so many different subjects, any given video will only appeal to a small percentage of them. But spread the word!
Thanks so much, helped a lot for my coming exame!! Greetings from Germany
Could you please make a video explaining RNA sequencing. Thanks.
I thought this was some advanced connect4 technique.
that was so helpful thanks prof😉
Thanks for the info you made it see easy
Wont you use q pcr for expression?
you are a legend
Great
He looks like a smarter version of Charlie day ❤️
Sir but how can this be used for quantitative assay of a particular gene...i mean i only understand that this will be only useful for qualitative analysis
the general idea: with each pcr cycle, the amount of a certain DNA sequence is double. Using some kind of labeling, e.g. fluorescent dyes, we can detect the presence of the mentioned DNA sequcense. The fluorescent dye has a detection limit (minimal concentration of the substance which can be detected), so, if we know that we detected the DNA after, let's say, 9 PCR cycles, and the detection limit for the dye is 100 nmol/ml, that means that the amount of DNA in the initial sample was 2^9 times lower, thus, quantitative analysis.
Hey Dave, just a quick question. How would the cDNA bind to the DNA in microarray if the primary transcript formed after trancsription is spliced? you can just link me some resources for the answer if you want to because i have been watching your videos and you seem to have quite a busy schedule.
C DNA binds to DNA in microarray due to phenomenon called complimentary base paring,
or we can say that A base always bind to T like that all base pairs are bound to attach to their complimentary strand due to all complimentary bsepairs . If i am wrong correct me.
@@shrikantnalkar4751 no i get that... i am aware of complementary base pairing.. my question was if the transcript of DNA goes under splicing, how does the cDNA bind to oligonucleotides on microarray... turns out oligonucleotides are the mRNA from which the cDNA is made and not a piece of DNA in the microarray... basically my doubt is solved after doing some research but thnx anyway
👍👍👍💙
Am i the only one listening to a different language here wtf 😳
Didn't know there exists a word called assay.
Why can’t my lecturers explain things simply like that?🙄
The first comment 😁
Wow
Oh man, the Professor with the cringy intro again..
I just wanted to give a heartfelt thank you for this video. My first year final tomorrow is an essay about using microarrays to identify heterochromality genes in huskys and this helped me study for it tremendously.
During the process of RT-PCR we get a complimentary ssDNA for which we perform PCR to get a dsDNA, we add this to each of the wells of the array plate and the complimentary DNA probe hybridizes and form a link. My doubt is if the DNA is already ds post RT-PCR how will it bind to another DNA probe. Or are we considering the DNA to be ss post RT- PCR. (the animation does not support the fact that the DNA is ss post RT-PCR at 5:11 ) Anybody?
same question
Why getting a 90€ Biochemistry-Textbook when RUclips can help you to understand the topic in about 8min? :)
Thank you for your content!
It helped me a lot! :)
Dude seriously. I'm learning Biochemistry and required to use the Lehninger book that everyone says is so good but I honestly am learning so much more from RUclips and only refer to the book when I have to. I'm getting along just fine without it.
Thank you so much for the video! I have a question. Does the cDNA have to be denatured, like with NaOH or something, so that it can bind to the single-stranded probes in the array?
yes.
Thank you very much, you helped me understand much better.
Greetings from Argentina!
I'm a little confused that the approach is to make double-stranded DNA with DNA polymerase and then try and get it to apparently separate and bind to single-stranded probes. Do the probes test for both the sense and antisense strands on each spot?
Same issue here , couldn't manged to understand that point.
Hey, you might already have found the answer to your question but in order to make cDNA we use a reverse transcriptase. This is a protein that basically turns your RNA back into DNA. Usually the reverse transcriptase leaves you with a single stranded DNA strand and a round of normal PCR (polymerase chain reaction) is required to turn it into double stranded DNA. Hybridization can then be performed by separating the strands again (either heating or chemical, where heating would be preferred since it is less harmful for the DNA). The stand that is complementary to the probe that was designed will then bind to either one of the strands and start the microarray assay. Which strand binds should not matter too much since the probe should be specific for a certain gene. Hope this helps.
This is great: super simple, super precise, it makes you really understand the basis of microarrays. From here you can go to more complex videos, but you do need a basis like this to start! Congratulations
Professor your explanation is informative, precise and beautiful thank you
So for microarray, we are only using one cDNA strand which was made from the template, the mRNA. However, there is one thing that isn't clear to me and that is about the RT-PCR. RT-PCR generates double-stranded cDNA right? How come in microarray we are only using the single strand and not double? Is there any intermediate enzyme that was involved?
thank you for everything you do!! :)
The explanation was excellent !! i used it to explain the DNA chips for my students
Thank you! One Question: How the RNA splicing during post-transcriptional modification does not cause any problems for RT-PCR?
In a nutshell, RNA splicing involves the removal of introns (non-coding parts of the mRNA) and joins the coding sequences (exons) together in order to enable translation. Since the parts removed are non-coding, it does not affect RT-PCR. RT-PCR, standing for Reverse Transcription Polymerase Chain Reaction, involves reverse transcriptase - converting RNA into cDNA - and (often) Taq DNA polymerase - completing the DNA after denaturation. To sum up, since the RT-PCR process only duplicates CODING DNA, DNA splicing does not affect the process AT ALL, since it removes NON-CODING parts of the original DNA.
Thanku very much professor your video really helped me in understanding microarray the way u explains is best
This was so amazing...but not gonna lie , the lizard freaked me out a little bit.
How have you not already made a connect four?
you sir have just saved my grade, i salute you o7
good work! thanks from austria!
I have a question
How could you use Gene Expression Arrays to identify alternative splicing
Some researchers have used algorithms to study expression differences between samples.
pubmed.ncbi.nlm.nih.gov/11435406/
thank you science jesus
Many thanks for this explanation professor Dave!
Thank you so much for the effort you put into this, its was so easy to understand
I fucking love you 😭
Could someone explain, how do we know where the complementary strands in the microarray came from (as in, from cell type 1 or cell type 2)?
Thanks, I hope you make video about RNA microarrays, it will be so completable !
I really like how you explain the science. I like these video.
YOUR LESSON IS ATTRACTIVE AND GUIDELINE FOR US. BUT I HAVE A COMMENT. HOW CAN I CONTACT YOU PERSONALLY?
Using this bc I want to better understand what I’m selling lmao. I’m a bio pharma sales rep!
why don't we extract the DNA instead of extracting the mRNA and then reverse it to cDNA ? i don't get it what is the point from this?
Totally dig your style and material! Go Science and kick some assays..
ohhh you have remove hair.. Quarantine trick..?? Nice
Is this this DNA microarray process described in the video also known as "bulk" RNA sequencing?
Thanks. I watched it just for general interest in this subject.
DUMB QUESTION: why can't the original DNA with the gene be isolated from the beginning? Why must the mRNA be isolated first, just to then be reverse engineered into the original DNA?
The way I understand it, not all of the genes present in the DNA, are being expressed/transcribed. So in order to only identify the currently transcribed genes, you have to work with mRNA.
Thank you very much , Professor ❤❤❤❤❤❤🙏🙏🙏
Amazing video👌👌👌. It was really helpful. Now the concept is clear for me. Thank you soo much for this video 😃.
med student here. wish our professors can teach like this...
please sir discribed the discovery of drug through micoarray
thankyouu😌 I never understood microarrays in my class
omg. You are so talented man, the more I grow up, the more I realize how genius and knowledgeable you are. Cheers to you from Saudi Arabia.
100000 times better than my textbook
My question is mRNA already bound and complementary cDNA is formed then how they bind with dna probes present in wells
word of caution, rna pol doesnt need a primer right, so reverse transcriptase having a primer feels fishy but its not because although widely called reverse trasncriptase the enzyme itself is synthesizing dna and so in essence its a dna pol and dna pols need the TTT primers.
Awesome! Thank you.
Wonderful presentation and thankyou 👌
Thank you prof Dave... Much love!!!
Nice!
I wish you could see what im seeing people!
I wish you could be here with me!!!
oooohh Baby HAHA!
BRING IT OOOONNN!
AND REMEMBER FOLKS!
YOU HEARD IT FIRST FROM JOHNYYYYAAAHHHHAHA! *dead*
I sent you email but you never replied.👌👌👌😢😢😢
sorry it may have gone to spam or it may have been something i couldn't answer!
@@ProfessorDaveExplains It was a physics question of class 9. I am sure that you are that much capable.
great haircut
Does this mean that we need to have a full genome sequenced of that specific organism in order to construct the microarray plate?
What kind of sequencing can you perform that Dave explains at 7:15? can you do Next gen sequencing?
Short hair dave is so fine
I FREAKING LOVE YOU PROFESSOR DAVE EXPLAINS!!!!!
Really helpful, Professor!
How is the double stranded cDNA "split" into a single strand to hybridize with the probes in the microarray?
An RNAse endonuclease would have to disassemble the RNA molecule from the cDNA, to allow for the hybridisation
Heating, same as for PCR with DNA polymerase
Very impressive explanation.
❤❤ thank you
Thank you prof
so you mean a common pool with all the different samples is allowed to hybridise on the array
@5:26 Do I have this right? (no background in science, just leaping in)
-There's a double stranded cDNA: one is identical to the DNA template and the other is complementary to template
--These two strands are bound together, right?
-"One of the two strands of some cDNA molecule made from an mRNA ought to bind to the DNA fragment in a particular well"
--How does one of the strand bind to the DNA fragment? My assumption is that the cDNA are bound to each other, so how does one of them separate and bind to the fragment?
Same as when you do PCR in the other direction: heat it
Thank you Professor Dave 😃
YOU ARE THE BEST
The genes should be expressed in 3D. As we are 3Dsal beings.
That doesn't mean anything.
VERY NICE
Thank you please more things explain about genotype
Amazing !
Hats off ^_^
great work! so much helpful in my Master's