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C-MYC AND CYCLIN D1 †/ RT-PCR


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#1 vickie

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Posted 22 May 2002 - 05:07 PM

I saw a decrease in †c-myc and cyclin D1 proteins expression †level in WB (related to control) †. But when trying to see the effect in RNA level using RT-PCR method I can not see any changes in the expression, and I donít know why. †
I know that the inhibitory affect I see is in the transcripional level (and not post translation).

Thank you for your help

vickie


#2 Elzbieta Janda

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Posted 24 May 2002 - 07:21 AM

The differences in mRNA probably are not so big to be detectable by a plain RT-PCR that even at 20 cycles can go to saturation .  If your protein level is clearly down- regulated why do you want to try RT-PCR?  I would not worry, but if you really have plenty of spare time try a semiquantitative RT-PCR or TaqMan PCR quantification.  

#3 scogne

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Posted 25 May 2002 - 01:25 AM

Hi,

as said before, your PCR maybe saturated. So try to determine a earlier stage were it will not be. (u can try ie: 20, 25 and 30 cycles or less... it depends on the srenght of expression.)
Good luck.





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