Real time PCR

[roniadam]

Hi:
Can anyone explain to me how SYBR green PCR can be used to determine DNA concentration ?

Second thing:
How are Reverse-Transcription PCR and Real-Time PCR combined to perform expression studies?

[MrMistery]

for number one:
The RT-PCR machine is equipped with a spectophotometer that will detect the concentration of DNA-SYBR green complex(i am assuming SYBR green is the DNA binding florocrome in the mix, i never asked myself what it is called) in your machine. Since the DNA concentration increases exponentially in the PCR reaction, the line showing the concentration will shoot up at one point. Depending on how quickly it shoots up, you can deduce how much DNA was there to begin with.

for number two
You can extract mRNA from the cell, do reverse transcription with polyU primers to turn it all into cDNA and then do RT-PCR with gene-specific primers to find out how much mRNA for that particular gene you originally had. It is fast and reliable, much easier than a Northern Blot.

Cheers

[canalon]

A really good ressource on the subject is this one:

http://pathmicro.med.sc.edu/pcr/realtime-home.htm

That should answer all of your questions.

[hadis7]

Hi,
can anyone explain why i can't have a good standard curve and it has a lot of variation?

[bravebeaker]

make sure to vortex your DNA samples before you mix them for the qPCR in order to generate the std curve. also make sure you measured the DNA accurately to make the std curve calculation, for example I found out that checking by nanodrop doesnt give me good results as good as Qubit (different measurements) but I believe Qubit is more accurate.
By the way how do you prepare your std curve exactly? How many dilutions do you make? What kin of cycler do you use?