real-time PCR - (Jun/03/2005 )
Hy dear friends!
I obtained good real time results when I make a nested with the same mix with 2.5 microliter of first real time PCR..........., why this happened?
thanks
Hi,
how big is the PCR product in your first PCR? Ideally, the size of amplicon is between 60 to 150bp. A smaller product could improve your results significantly.
I am not very experienced in rt-PCR, it's just an idea.
Cheers,
Freiberger
Hi mariateresav,
What do you mean by good real-time result? In what way your nested PCR improved your real-time result?
Is your Ct value become smaller, fluoresence value become higher, increase sensitivity (pick up less copy of DNA), your standard curve become more consistance?
How is your real-time result before nested PCR?
Thank
Hadrian
What do you mean by good real-time result? In what way your nested PCR improved your real-time result?
Is your Ct value become smaller, fluoresence value become higher, increase sensitivity (pick up less copy of DNA), your standard curve become more consistance?
How is your real-time result before nested PCR?
Thank
Hadrian
yes my sensitivity was increased because in the first PCR I detected nothing..........., in nested, same mix with same primers..............my negative control resulted nothing again but my samples increased fluorescence with small Ct value...........,
thank
thank
Dear mariateresav,
This is not surpricing as you had introduced more template into your second PCR.
You might have very little amount of total DNA in your sample. Thus your first real-time PCR is not able to detect a sigmoidal signal. However your specific PCR product had increased through out your first PCR. So when you proceed to second PCR, you actually introduced more DNA inside as initial template. Thus you will see your real-time is result improved.
Generally, real-time PCR need at least 10 pg DNA to be detected.
Are you quantitate your DNA sample prio real-time PCR?
You should do that to ensure you work quality.
Can I know what sample are you dealling with and what extraction methods you are using?
When we say something had improved my real-time result, it means it increased:
1) PCR effeciency,
2) specificity (higher fluoresence signal, lower background)
3) detection limit.
Hope my explanation can help you in understanding more about real-time PCR.
Best regard
Hadrian.
Dear mariateresav,
This is not surpricing as you had introduced more template into your second PCR.
You might have very little amount of total DNA in your sample. Thus your first real-time PCR is not able to detect a sigmoidal signal. However your specific PCR product had increased through out your first PCR. So when you proceed to second PCR, you actually introduced more DNA inside as initial template. Thus you will see your real-time is result improved.
Generally, real-time PCR need at least 10 pg DNA to be detected.
Are you quantitate your DNA sample prio real-time PCR?
You should do that to ensure you work quality.
Can I know what sample are you dealling with and what extraction methods you are using?
When we say something had improved my real-time result, it means it increased:
1) PCR effeciency,
2) specificity (higher fluoresence signal, lower background)
3) detection limit.
Hope my explanation can help you in understanding more about real-time PCR.
my template was cDNA from very little amount of serum RNA....... it was undetectable by spectofotometer.......
your explanation was very useful for me!
thank very much!!!!
Best regard
mariateresav