problem with b-actin CT values - (Oct/17/2006 )

Hi,

I'd like to ask for some help, 'cause the RT-PCR drives me crazy. I want to measure the down regulation efficiency but everytime when I perform a RT I can not use it because there is a huge fluctuation in the Ct values of actin(16-22). I tried several things like random primers, oligo dT, M-MLV Rt, SSII and so on. I purify the RNA with Trizol, and according to the Nanodrop it looks OK. But still.
I never have nice results my curves look terrible and I am out of new ideas. I've already ordered GAPDH primers to test them, but I don't think that it'd help.
Somebody any idea?
Thanks a lot!!!

What kind of system are you using? I found that using a Universal Master Mix (Applied Biosystems) containing everything except primers and probes for all of my samples greatly increased the consistency of my Ct values from run to run.

Assuming you are using ABI primer-probe (AoD). Use Qiagen RNeasy Spin-Column to purify your RNA, rather than Trizol. The rational is this: little phenol could cause you higher 260 OD, consequently, higher than expected RNA amount. Slight contamination of phenol from trizol will affect the Reverse Trascription. Hope this may remove issues that you have with fluctuating Ct for b-actin. Please post your primer-probe sequence if not from ABI. Thanks.

yeah, i agree with cixlar, you can't really use trizol as the salt concentration alone will interfere with your real-time... use a membrane/spin based system, qiagens is good, promegs SV total rna isolation kit is cheaper with comparable results

Hi guys,

Thanks for the help. Maybe it is good idea to try other methods for RNA isolation. In the lab we have invitrogen, bio-rad and qiagen kits so I will try one of these. For the PCR we use preprepared Sybrgreen from Invitrogen we just add the primers and water. I just found out that our pipetting robot could also be responsible for this problem because it is not really accurate.
I tested my newly ordered GAPDH primers and the result is the same but at this time my duplicates were not really identical either I had a difference of 1.5 cycle which is impossible
I try it again