Realtime PCR standard curves - (Jun/02/2005 )
Hello
After quite a few trials I got a decent regression value, yet the slope is very poor about 1.2. what could have gone wrong?
It would be great if any of you have any suggestions!!!
b
One thing you should check is that you RNA extraction. If there are a lot of salt during your extracting procedure, it will interfer with the Taq polymerase that will change you amplicafication ability. So the ratio could not be aroung -3.32.
Dear Abi,
Sorry, I get you wrong just now, sorry 
You are in right forum.
Can I know what real-time probe are you using, is TaqMan or SYBR Green?
Thank
Hadrian
Hello
I am using SyBr Green for detection.
Are there any suggestions to improve the slope and thus the efficiency of my real time PCR assay
Regds
Abi
Hello yuelongshan
I do a Trizol extraction for isolating total RNA. I am not sure as to how to address the salt concentration. Any thoughts on that?
I do a Trizol extraction for isolating total RNA. I am not sure as to how to address the salt concentration. Any thoughts on that?
Hi,
what kind of tissue are you using? Maybe i have a good protocol for it?
I use C.elegans whole animals.
Sorry, i don't have protocol for it.
Make sure that you only add 10% of the RT reaction into pcr reaction due to the inhibitors.
Optimize your primers too since you are using Syb green as the dye. THe length of the product ideally must be 80-150bp for efficient amplification.
Dear Abi,
Since your slope is 1.2 taht mean your real-time reaction is not carry out in an exponential way. In another word your primer efficiency is low.
two reasons can contribute to this situaition, 1) sub-optimal primer design or PCR condition. 2) extraction method.
From my experience, TRIzol extraction method is not suitable for real-time PCR application. It contain excessive salt which make your template deficult to denature during denaturation. Try to use column base purification kit. BioNeer purification kit is a good one.
You can also run a gel after your real-time PCR. If you can see a single band without smearing effect, that means your primer design and PCR condition is ok. so the problem will be your extraction method.
Good try.
Hadrian